Application of targeted Hsp90 alpha compound in treatment of infectious encephalitis and improvement of clinical drug resistance

Abstract

The invention provides application of a targeted Hsp90 alpha compound in treatment of infectious encephalitis and improvement of clinical drug resistance, and belongs to the technical field of medicines. The targeted Hsp90 alpha compound disclosed by the invention has a structure as shown in a formula 1, and a parent nucleus structure of the targeted Hsp90 alpha compound is (3, 6, 6-trimethyl-4-oxo-4, 5, 6, 7-tetrahydro-1H-indazole-1-yl) benzamide. The targeted Hsp90 alpha compound can effectively treat infectious encephalitis, can treat infectious encephalitis caused by acyclovir and other nucleoside drug resistance viruses, and improves the clinical drug resistance problem of infectious encephalitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a compound targeting Hsp90α in the treatment of infectious encephalitis and in improving clinical drug resistance. Background Technology

[0002] Infectious encephalitis is an inflammatory response caused by the direct invasion of brain tissue by pathogens, most commonly seen in herpes simplex virus (HSV-1) encephalitis. Its main pathogenic mechanism is that HSV-1 establishes latent infection in trigeminal ganglion cells to evade the body's immune system, leading to lifelong HSV-1 infection and triggering herpesvirus encephalitis. Currently, commonly used antiviral drugs are mainly nucleoside analogs and their analogues, represented by acyclovir. These drugs inhibit viral genome replication through competitive action with normal deoxyribonucleoside triphosphates. However, long-term use leads to severe drug resistance and does not reduce brain inflammation. Therefore, there is a lack of long-term effective treatments for HSV-1 encephalitis. Summary of the Invention

[0003] The purpose of this invention is to provide an application of a Hsp90α-targeting compound in the treatment of infectious encephalitis and in improving clinical drug resistance. The Hsp90α-targeting compound of this invention can effectively treat infectious encephalitis and improve the clinical drug resistance problem in infectious encephalitis.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis, wherein the Hsp90α-targeting compound has the structure shown in Formula 1:

[0006]

[0007] In Formula 1, R is selected from amino heterocyclic groups.

[0008] This invention provides the application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis caused by nucleoside analogue-resistant viruses, wherein the Hsp90α-targeting compound has the structure shown in Formula 1:

[0009]

[0010] In Formula 1, R is selected from amino heterocyclic groups.

[0011] Preferably, the Hsp90α-targeting compound has the structure shown in Formula 1-1, Formula 1-2, or Formula 1-3:

[0012]

[0013] Preferably, the Hsp90α-targeting compound targets and binds to the Hsp90α protein, and the binding site between the Hsp90α-targeting compound and the Hsp90α protein is LYS58.

[0014] Preferably, the infectious encephalitis is herpesvirus encephalitis.

[0015] Preferably, the herpesvirus encephalitis is herpesvirus encephalitis caused by HSV-1.

[0016] Preferably, the infectious encephalitis treatment drug activates the body's TRIM56-cGAS-STING immune pathway through the Hsp90α-targeting compound, promotes I-IFN production, and enhances the body's immune function to treat herpesvirus encephalitis caused by HSV-1.

[0017] Preferably, the nucleoside analogue includes acyclovir.

[0018] Preferably, the dosage form of the drug for treating infectious encephalitis includes oral preparations, topical preparations, or injectable preparations.

[0019] Preferably, the oral preparations include tablets, capsules, pills, sugar-coated preparations, sustained-release preparations, controlled-release preparations, or suspensions; the topical preparations include lotions, creams, gels, ointments, microneedles, sprays, or drops; and the injectable preparations include injection solutions or powder injections.

[0020] This invention provides the application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis. The Hsp90α-targeting compound has the structure shown in Formula 1, with its core structure being (3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide. The Hsp90α-targeting compound of this invention can effectively treat infectious encephalitis, and can also treat infectious encephalitis caused by nucleoside analogue-resistant viruses such as acyclovir, thus improving the clinical drug resistance problem in infectious encephalitis. Specifically, the Hsp90α-targeting compound of this invention enhances the ubiquitination function of TRIM56 by binding to the Hsp90α protein after HSV-1 virus infection, quantitatively regulating the increase in type I interferon (I-IFN) expression, thereby exerting anti-HSV-1 virus efficacy. Meanwhile, the Hsp90α-targeting compound of the present invention can solve the clinical drug resistance problem of infectious encephalitis, and the Hsp90α-targeting compound also has the effect of enhancing the body's I-IFN compared with acyclovir, a commonly used drug for the clinical treatment of infectious encephalitis. Attached Figure Description

[0021] Figure 1 The graph shows the CCK8 cytotoxicity test results for compounds JD13 and JD10.

[0022] Figure 2 The image shows the results of the plaque assay for compound JD13.

[0023] Figure 3 The figure shows the results of the expression test of IFN-related factors of compound JD13 on Ifnβ, Cxcl10 and Isg15;

[0024] Figure 4 The graph shows the results of in vitro I-IFN expression level determination for different compounds;

[0025] Figure 5 The image shows the results of in vitro HSV-1 infection detection of Hsp90α protein expression.

[0026] Figure 6 Figure showing the validation results for the effect of knocking down Hsp90α on EGFP-HSV-1 infection;

[0027] Figure 7 The graph shows the results of molecular dynamics simulations of compounds JD13 and Hsp90α.

[0028] Figure 8 The diagram shows the molecular docking results of compound JD13 and Hsp90α.

[0029] Figure 9 The figure shows the results of a cell heat transfer assay showing the specific binding of compound JD13 to Hsp90α.

[0030] Figure 10 This is a graph showing the plaque validation results of compound JD13 against HSV-1 infection.

[0031] Figure 11 The image shows the results of detecting Hsp90α protein expression induced by HSV-1 infection in vivo.

[0032] Figure 12 The figure shows the results of in vivo detection of compound JD13 on symptoms of herpesvirus encephalitis and the expression of HSV-1 gene copy numbers UL54, UL52, and UL27 in brain tissue;

[0033] Figure 13 The figure shows the results of detecting the effect of Hsp90α knockdown on HSV-1-induced INFβ secretion levels.

[0034] Figure 14 The graph shows the verification results of the interaction between compound JD13 and cGAS and TRIM56 via Hsp90α.

[0035] Figure 15 The figure shows the verification results of the binding of compound JD13 to cGAS and viral DNA via Hsp90α.

[0036] Figure 16 The figure shows the detection results of compound JD13 activating the upstream protein of I-IFN via TRIM56. Detailed Implementation

[0037] This invention provides the application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis, wherein the Hsp90α-targeting compound has the structure shown in Formula 1:

[0038]

[0039] In Formula 1, R is selected from amino heterocyclic groups.

[0040] This invention provides the application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis caused by nucleoside analogue-resistant viruses, wherein the Hsp90α-targeting compound has the structure shown in Formula 1:

[0041]

[0042] In Formula 1, R is selected from amino heterocyclic groups.

[0043] In the above formula 1 of the present invention, R includes, but is not limited to, (4-hydroxybicyclo[2.2.2]octane-1-yl)amino, (5-hydroxyadamantane-2-yl)amino, or 4-acetyl-3-(piperidine-4-amino)phenyl.

[0044] As one embodiment of the present invention, the Hsp90α-targeting compound has the structure shown in Formula 1-1, Formula 1-2, or Formula 1-3:

[0045]

[0046] The core structure of the Hsp90α-targeting compound of the present invention is (3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole-1-yl)benzamide, wherein the functional group of the Hsp90α-targeting compound having the structure shown in Formula 1-1 is 2-((4-hydroxybicyclo[2.2.2]octan-1-yl)yl), and its chemical name is 2-((4-hydroxybicyclo[2.2.2]octan-1-yl)yl)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole-1-yl)benzamide, abbreviated as compound JD13; the Hsp90α-targeting compound having the structure shown in Formula 1-2 is abbreviated as compound JD10; and the Hsp90α-targeting compound having the structure shown in Formula 1-3 is abbreviated as compound JD14.

[0047] In one embodiment of the present invention, the Hsp90α-targeting compound targets and binds to the Hsp90α protein, and the binding site of the Hsp90α-targeting compound to the Hsp90α protein is LYS58. The Hsp90α-targeting compound of the present invention can target Hsp90α, promote the activation of cGAS in the body by targeting and binding to Hsp90α, enhance the binding ability of cGAS to viral DNA, thereby promoting the production of I-IFN in the body, improving antiviral innate immune efficacy, and thus exerting a therapeutic effect on infectious encephalitis.

[0048] In one embodiment of the present invention, the infectious encephalitis is herpesvirus encephalitis; the herpesvirus encephalitis is herpesvirus encephalitis caused by HSV-1.

[0049] In one embodiment of the present invention, the infectious encephalitis treatment drug activates the TRIM56-cGAS-STING immune pathway in the organism through the Hsp90α-targeting compound, promoting I-IFN production and enhancing the body's immune function to treat herpesvirus encephalitis caused by HSV-1. The Hsp90α-targeting compound of the present invention, by inhibiting Hsp90α, can activate the TRIM56-cGAS-STING immune pathway in the organism, promoting I-IFN production and enhancing the body's immune function, thus exerting a therapeutic effect on herpesvirus encephalitis caused by HSV-1, which is beneficial for the early therapeutic application of the drug in viral infections.

[0050] In one embodiment of the present invention, the nucleoside analogue includes acyclovir. The Hsp90α-targeting compound of the present invention can solve the clinical drug resistance problem in infectious encephalitis, and compared with acyclovir, a commonly used drug for the clinical treatment of infectious encephalitis, the Hsp90α-targeting compound also has the effect of enhancing the body's I-IFN.

[0051] As one embodiment of the present invention, the dosage form of the drug for treating infectious encephalitis may include oral preparations, topical preparations, or injectable preparations; the oral preparations may include tablets, capsules, pills, sugar-coated preparations, sustained-release preparations, controlled-release preparations, or suspensions; the topical preparations may include lotions, creams, gels, ointments, microneedles, sprays, or drops; and the injectable preparations may include injection solutions or powder injections.

[0052] In one embodiment of the present invention, the infectious encephalitis treatment drug includes an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes a compound targeting Hsp90α; the present invention does not specifically limit the type of pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients known to those skilled in the art can be used.

[0053] Studies show that I-IFN is a factor in antiviral immunity and the regulation of immune responses. During infection, I-IFN is produced in large quantities, enhancing the body's immune response and resisting DNA and RNA viral infections. Therefore, whether a drug can balance I-IFN production in both resting and infected states is a key indicator for evaluating the efficacy of drug treatment for infectious encephalitis. Hsp90α is a stress-induced subtype of the molecular chaperone Hsp90, mainly existing in the cytoplasm as a homodimer (αα). Under stress conditions (such as viral infection), Hsp90α is induced to express and assists proteins such as protein kinases, transcription factors, and the E3 ubiquitin ligase TRIM56 in correct folding, maintaining stable conformations, assisting in precise protein localization and efficient protein complex assembly, thereby exerting the therapeutic effect of drugs on infectious encephalitis. Based on this, the present invention provides the application of Hsp90α-targeting compounds in the preparation of drugs for the treatment of infectious encephalitis, and evaluates their antiviral and innate immune efficacy. Specifically, it verifies their binding ability to Hsp90α, determines that they are Hsp90α-targeting proteins, inhibit the function of Hsp90α during infection, activate E3 ubiquitin ligase TRIM56 to promote the anti-infective effect of I-IFN in the body.

[0054] In this embodiment of the invention, the binding of the Hsp90α-targeting compound (compound JD13) and other compounds with similar core structures (compound JD10, compound JD14, the specific structures of which are shown in Table 1) to the Hsp90α target protein after viral infection, the TRIM56 ubiquitination function, and the I-IFN expression were studied to evaluate the therapeutic effect of the drugs in vitro and in vivo.

[0055] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] Example 1: Anti-infective activity and in vitro toxicity verification of three compounds with (3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide as the parent core.

[0057] The experimental method is as follows:

[0058] (1) Culture Vero cells in an appropriate culture medium, seed them in 96-well plates at a rate of 10,000 cells / well, and place them in a cell culture incubator at 37°C and 5% CO2 to allow them to adhere to the plate overnight.

[0059] (2) Take appropriate amounts of the test compounds (different compounds with the parent structure of (3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole-1-yl)benzamide, namely compound JD13, compound JD10, and compound JD14, the specific structures of which are shown in Table 1) and perform serial dilutions of 2-fold using cell maintenance medium to obtain a series of compound solutions with different concentrations (at this time, the concentration of the diluted compound should be twice the final concentration of the desired compound). Then, calculate the amount of virus and the dilution according to MOI = 0.1. The formula for calculating the amount of virus is: amount of virus (V) = (MOI × number of cells per well × number of wells) / (PFU / mL), PFU / mL = TCID50 × 0.7.

[0060] (3) Observe and record changes in cell morphology and statistically analyze the inhibitory effect of different compounds on cell pathogenesis; the normal group consists of cells cultured in cell maintenance medium, and the virus infection group consists of cells infected with virus at MOI=0.1 diluted in cell maintenance medium.

[0061] (4) Compounds JD13 and JD10 were selected and their toxicity to microglia was determined using the CCK-8 assay to determine the maximum non-toxic concentration (TC0). The test compounds were then diluted in a serial manner and inoculated into microglia. After culture, the cell viability was measured.

[0062] Experimental Results: The anti-infective activities of different test compounds are shown in Table 1. It can be seen that all three compounds with the core structure (3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazole-1-yl)benzamide exhibit anti-HSV-1 infection activity. Compound JD13 showed relatively good anti-infective activity at a low dose (0.25 μM). Compound JD10 showed good anti-infective activity at a medium dose (0.5 μM). Compound JD14 showed good anti-infective activity at a high dose (1 μM).

[0063] Figure 1 The graph shows the CCK8 cytotoxicity test results for compounds JD13 and JD10. The results indicate that compound JD13 has lower cytotoxicity and is more valuable for clinical drug development.

[0064] Table 1. Inhibitory effects of different compounds on HSV-1-infected cytopathic effects.

[0065]

[0066] Note: In Table 1, no cytopathic effect after infection is marked as "-"; 1-25% cytopathic effect is marked as "+"; 26-50% cytopathic effect is marked as "++"; 51-75% cytopathic effect is marked as "+++"; and 76-100% cytopathic effect is marked as "++++".

[0067] Example 2: Using compound JD13 as an example to verify the efficacy in treating acyclovir (ACV) resistant virus strains.

[0068] The experimental method was as follows: Vero cells were seeded into 24-well plates at a density of 350,000 cells / well, with 500 μL per well, to ensure that the cells could grow into a uniform monolayer after overnight culture. The next day, compound JD13 was co-treated with normal HSV-1 / F and ACV-resistant strains HSV-1 / 153 and HSV-1 / Blue for 2 h, respectively. The medium was then replaced with 1 mL of maintenance medium (2×DMEM: 1% methylcellulose solution = 1:1, volume ratio) and cultured for 72 h. Afterward, the cells were fixed with 4% paraformaldehyde for 20 min, stained with crystal violet for 30 min, and then rinsed with water. Finally, the number of plaques was counted to evaluate the effect of compound JD13 on HSV-1 and the resistant strains.

[0069] Experimental results: Figure 2 The figure shows the results of the plaque assay for compound JD13. The results show that compound JD13 can inhibit the number of plaques formed by normal virus strain HSV-1 / F and drug-resistant ACV strains HSV-1 / 153 and HSV-1 / Blue in a dose-dependent manner, and the inhibition rate of compound JD13 against the three strains is over 95% at a concentration of 100 nM.

[0070] Example 3: Measurement of the expression levels of cellular immune factor I-IFN and its related factors Ifnβ, Cxcl10 and Isg15 by in vitro drug binding to target protein Hsp90α.

[0071] The expression of IFN-related factors such as Ifnβ, Cxcl10, and Isg15 by compound JD13 was detected by RT-qPCR. The specific experimental method is as follows: Microglia BV2 cells were seeded at 350,000 cells / well in 6-well plates and cultured overnight in a 5% CO2 incubator at 37°C. The next day, the cells were transfected with the appropriate reagents (transfection buffer, serum-free DMEM) and 4 μg of Hsp90α plasmid, vortexed for 10 s, and centrifuged for 2 s. The transfection complex was then incubated at room temperature for 10 min, and then added dropwise to the culture dish using a pipette tip while slowly mixing the liquid to ensure even distribution. After 6 h of transfection, the medium was replaced with 10% FBS in complete DMEM medium, and the cells were cultured for another 48 h before subsequent experiments. Cells overexpressing Hsp90α were cultured with HSV-1 (MOI) 1) After infection and co-treatment with compound JD13 for 12 h, the supernatant was discarded, and the cells were washed once with PBS and the supernatant was discarded again. Cells were then lysed with 1 mL Trizol, followed by the addition of 200 μL chloroform and centrifugation at 4 °C and 12,000 rpm for 15 min. After centrifugation, the supernatant was transferred to a new EP tube, and an equal volume of isopropanol was added. The cells were then centrifuged at 4 °C and 12,000 rpm for 10 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in 500 μL of 75% ethanol (75 mL of ethanol was added to 25 mL of DEPC water). The cells were then centrifuged at 4 °C and 12,000 rpm for 5 min. Finally, the supernatant was discarded, and an appropriate amount of DEPC water was added to dissolve the precipitate. The extracted brain tissue RNA sample was added to reverse transcriptase, and 2000 ng of cDNA was reversed. Then, RT-qPCR was used to detect the expression of IFN-related factors Ifnβ, Cxcl10, and Isg15 by compound JD13. The primers and their corresponding sequences involved in the RT-qPCR experiment are shown below:

[0072] Ifnβ-F: 5'-ATGAGTGGGTGGTTGCAGGC-3' (SEQ ID NO. 1);

[0073] Ifnβ-R: 5'-TGACCTTTCAAATGCAGTAGATTCA-3' (SEQ ID NO. 2);

[0074] Cxcl10-F: 5'-CCAAGTGCTGCCGTCATTTTC-3' (SEQ ID NO. 3);

[0075] Cxcl10-R: 5'-GGCTCGCAGGGATGATTTTCAA-3' (SEQ ID NO. 4);

[0076] Isg15-F: 5'-GATTGCCCAGAAGATTGGTG-3' (SEQ ID NO. 5);

[0077] Isg15-R: 5'-TCTGCGTCAGAAAGACCTCA-3' (SEQ ID NO. 6);

[0078] β-actin-F: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID NO.7);

[0079] β-actin-R: 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID NO. 8).

[0080] Experimental results: Figure 3 The figure shows the results of the expression test of IFN-related factors of Ifnβ, Cxcl10 and Isg15 by compound JD13. The results show that the expression of IFN-related factors of Ifnβ, Cxcl10 and Isg15 is significantly increased, suggesting that compound JD13 can promote the increase of antiviral factor secretion in the body after binding to the target protein.

[0081] Comparative Example 1: Determination of I-IFN Expression Levels in Acyclovir, a Commonly Used Clinical Drug, in Vitro

[0082] RT-qPCR was used to detect the expression of I-IFN factor by different compounds. The specific experimental method is as follows: Microglial cells (BV2) were seeded at 600,000 cells / well in 6-well plates and cultured overnight in a cell culture incubator at 37°C and 5% CO2. The next day, cells were infected with HSV-1 (MOI 1) and co-treated with compounds JD13, JD10, and ACV for 12 h. After the supernatant was discarded, the cells were washed once with PBS and the supernatant was discarded. Cells were then lysed with 1 mL of Trizol, followed by the addition of 200 μL of chloroform and centrifugation at 12000 rpm for 15 min at 4°C. After centrifugation, the supernatant was transferred to a new EP tube, and an equal volume of isopropanol was added. The cells were then centrifuged at 12000 rpm for 10 min at 4°C. After centrifugation, the supernatant was discarded, and 500 μL of 75% ethanol was added (75 mL of ethanol was added to 25 mL of ethanol). The sample was resuspended in DEPC-treated water (75% ethanol) and then centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in DEPC-treated water. RNA samples from brain tissue were extracted, and reverse transcriptase was added to reverse-engineer 2000 ng cDNA. RT-qPCR was then used to detect the expression of different compounds on the IFN factor. The primers and their corresponding sequences used in the RT-qPCR experiment are shown below:

[0083] Ifnβ-F: 5'-ATGAGTGGGTGGTTGCAGGC-3' (SEQ ID NO. 1);

[0084] Ifnβ-R: 5'-TGACCTTTCAAATGCAGTAGATTCA-3' (SEQ ID NO. 2);

[0085] β-actin-F: 5'-GGCTGTATTCCCCTCCATCG-3' (SEQ ID NO.7);

[0086] β-actin-R: 5'-CCAGTTGGTAACAATGCCATGT-3' (SEQ ID NO. 8).

[0087] Experimental results: Figure 4 The figure shows the results of in vitro I-IFN expression level determination of different compounds. Combined with the experimental results in Example 3, it can be seen that after microglia were infected with HSV-1 (MOI 1) and co-treated with ACV for 12 h, compound JD13 significantly promoted I-IFN expression compared to compound JD10, but ACV did not promote I-IFN expression. Therefore, compared to the clinically commonly used drug acyclovir, compounds with the core structure 2-((4-hydroxybicyclo[2.2.2]octane-1-yl)yl)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide, such as compounds JD13 and JD10, can enhance cellular immune function.

[0088] Example 4: Identification of key target proteins in HSV-1-infected cells

[0089] The expression levels of total Hsp90 protein and its subtypes Hsp90β and Hsp90α during HSV-1 (MOI 1) infection of microglia BV2 were determined by Western blotting. The effects of target proteins on viral infection were detected by siRNA transfection. The specific experimental methods are as follows:

[0090] (1) Microglia BV2 cells were seeded at 600,000 per well in 6-well plates and placed in a cell culture incubator at 37°C with 5% CO2 for overnight culture. The next day, the cells were infected with HSV-1 (MOI 1) for 24 hours, and protein samples were obtained by SDS lysis. The protein concentration was then determined by BCA protein assay. Specifically, 40 μg of protein was subjected to SDS-PAGE electrophoresis, and the PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. Then, it was incubated overnight at 4°C in the presence of specific antibodies. Finally, the expression levels of total Hsp90 protein and its subtypes Hsp90β and Hsp90α were determined by Western blotting during the HSV-1 (MOI 1) infection of microglia BV2 cells.

[0091] (2) Microglial cells BV2 were seeded at 350,000 per well in 6-well plates and placed in a cell culture incubator at 37°C with 5% CO2 to allow them to adhere overnight. The next day, the appropriate amount of siRNA-Hsp90α was diluted with the corresponding reagent (transfection buffer, serum-free DMEM), vortexed for 10 seconds, and centrifuged for 2 seconds using a handheld centrifuge. The appropriate amount of transfection reagent was added to the diluted siRNA mixture, vortexed for 10 seconds, and centrifuged for 2 seconds using a handheld centrifuge. The transfection complex was then allowed to stand at room temperature for 10 minutes (5 minutes for the siRNA mixture). After standing, the transfection complex was added dropwise to the culture dish using a pipette tip while slowly mixing the liquid in the plate to ensure even distribution. After 6 hours of transfection, the culture medium was changed to 10% FBS DMEM and cultured for 48 hours to obtain the Hsp90α cell line. Subsequently, the virus was infected with UV-inactivated EGFP-HSV-1 strain (Mock group) and EGFP-HSV-1 strain (MOI 1) for 24 h, and the changes in immunofluorescence were compared.

[0092] Experimental results: Figure 5 The figure shows the results of HSV-1 infection on Hsp90α protein expression in vitro. The results show that the protein expression levels of total Hsp90 protein and its subtype Hsp90β did not change significantly during HSV-1 (MOI 1) infection of microglia BV2, but HSV-1 infection significantly promoted Hsp90α protein expression. Figure 6The image shows the verification results of the effect of Hsp90α knockdown on EGFP-HSV-1 infection (scale bar: 200 μm). Specifically, after knocking down Hsp90α in BV2 microglia with siRNA for 48 h, the resulting Hsp90α cell lines were infected with UV-inactivated EGFP-HSV-1 virus (Mock group) and EGFP-HSV-1 virus (MOI 1) for 24 h. Immunofluorescence results showed that, compared with the EGFP-HSV-1 infection group, Hsp90α knockdown significantly inhibited the proliferation of the EGFP-HSV-1 virus. Therefore, Hsp90α is a major regulatory protein in the HSV-1 infection process.

[0093] Example 5: In vitro virtual computation for predicting the antiviral activity of compound JD13

[0094] The experimental method is as follows: Taking compound JD13 as an example, the molecular dynamics method (supplemented by molecular dynamics calculations) was used to calculate the binding relationship between compound JD13 and Hsp90α. The molecular docking method (supplemented by molecular docking calculations) was used to predict the interaction sites between compound JD13 and Hsp90α, and the number of hydrogen bonds was counted to determine the key binding sites.

[0095] Experimental results: Figure 7 The graph shows the results of molecular dynamics simulations of compounds JD13 and Hsp90α. Figure 8 The diagram shows the molecular docking results of compound JD13 and Hsp90α. The two columns of icons in the lower right corner represent: van der Waals bonds (top left), hydrogen bonds (middle left), and π-σ hydrophobic interactions (bottom left); π-π conjugation effects (top right), alkyl groups (middle right), and π-alkyl hydrophobic interactions (bottom right). The results show that molecular dynamics simulations and hydrogen bond number statistics reveal a stable bond between compound JD13 and Hsp90α. Molecular docking confirmed the existence of interaction sites between JD13 and Hsp90α, identifying LYS58 as the key site for their binding.

[0096] Example 6: Verification of the anti-infective efficacy of drugs in vitro by binding to the key target protein Hsp90α

[0097] The specific binding of compound JD13 to Hsp90α was determined using a cell heat transfer assay, and the antiviral activity of compound JD13 was determined using a viral plaque assay. The specific experimental methods are as follows:

[0098] 1) Spread microglia into 100mm culture dishes at a cell density of 5×10⁻⁶ cells / mL. 5 / mL, 8mL per well. On the second day, the control dish was replaced with cell maintenance medium. The compound JD13 group was treated with compound JD13 diluted with cell maintenance medium (2μM) and incubated at 37℃ in a 5% CO2 incubator for 3h. The culture medium was aspirated, and the cells were washed twice with PBS. After adding 1mL of PBS, the cells were gently scraped off with a cell scraper and collected in 1.5mL centrifuge tubes. The cells were collected by centrifugation at 3000rpm for 5min at room temperature. After centrifugation, the PBS was aspirated, and 400μL of PBS (containing a 1:50 ratio of protease inhibitor) was added to re-incubate the cells. The cells were suspended and aliquoted into PCR tubes, 40 μL per tube. The PCR instrument was set with a series of temperature gradients (37℃, 42℃, 47℃, 52℃, 57℃, 62℃), with each temperature gradient heating time being 3 min. The heated samples were subjected to three freeze-thaw cycles to fully lyse the cells. The samples were then centrifuged at 4℃ and 20,000 rpm for 20 min to remove insoluble proteins. After centrifugation, the supernatant was collected, mixed with 5× loading buffer, and heated at 100℃ for 10 min to lyse the cells. Finally, the expression levels of Hsp90α and the internal control were analyzed by Western blotting.

[0099] 2) According to 3.0×10 5 Vero cells were seeded into 12-well plates with 1 mL of culture medium per well. After cell adhesion (forming a dense, contiguous layer of cells) on the second day, the samples for the virus and compound JD13 were serially diluted, with 250 μL of the diluted sample buffer added to each well, and three replicates were performed for each concentration. After incubation at 37°C with 5% CO2 for 2 hours, the sample buffer was carefully aspirated using a pipette tip, and the cells were washed twice with PBS. 0.5 mL of cell covering medium (1% methylcellulose + 2×DMEM) was added. After further incubation for 72 hours, the covering medium was discarded, and the cells were washed twice with PBS. 500 μL of 4% paraformaldehyde solution was added as fixative for 20 minutes. The fixative was discarded, and the cells were washed twice with PBS. 500 μL of crystal violet staining solution was added, and staining was performed for 30 minutes. After staining, the cells were rinsed with tap water until clear empty spots appeared. After drying, photographs were taken and the number of empty spots was counted.

[0100] Experimental results: Figure 9 The figure shows the results of a cell heat transfer assay that showed the specific binding of compound JD13 to Hsp90α. The results indicate that compound JD13 targets Hsp90α. Figure 10 The image shows the plaque validation results of compound JD13 against HSV-1 infection. The results show that compound JD13 has antiviral activity. It is a novel Hsp90α inhibitor that can significantly target and inhibit the function of Hsp90α, thereby inhibiting viral infection.

[0101] Example 7: Verification of the anti-infective efficacy of drugs in vivo through the key target protein Hsp90α

[0102] The expression of Hsp90α in brain tissue during HSV-1 infection in vivo was determined by Western blotting, and the effect of compound JD13 on viral DNA copy number was detected by RT-qPCR. The specific experimental methods are as follows:

[0103] 1) Using HSV-1 (2×10 6 Mice were infected with PFU / mouse drops intranasally and compound JD13 was administered intraperitoneally. HSV-1-infected brain tissue was obtained on day 8 of HSV-1 infection. The brain tissue was then lysed with SDS lysis buffer containing 1% PMSF and 1% phosphatase inhibitor to obtain protein samples. Cells were lysed using SDS lysis buffer containing 1% PMSF and 1% phosphatase inhibitor based on Western blotting. Protein concentration was then determined using the BCA protein assay. Specifically, 40 μg of protein was subjected to SDS-PAGE electrophoresis. The PVDF membrane was blocked with 30 mL of 5% skim milk (1.5 g of skim milk powder was dissolved in 30 mL of PBS solution containing 30 μL Tween 80) at room temperature for 1 h, and then incubated overnight at 4 °C in the presence of specific antibodies. Finally, the expression of Hsp90α in the brain tissue of HSV-1-infected mice was determined by Western blotting.

[0104] 2) Using HSV-1 (2×10 6 Eight days after mice were infected with PFU / mouse drops via nasal drip, brain tissue samples were ground with 1 mL of Trizol, followed by the addition of 200 μL of chloroform and centrifugation at 4°C and 12,000 rpm for 15 min. After centrifugation, the supernatant was transferred to a new EP tube, and an equal volume of isopropanol was added, followed by centrifugation at 4°C and 12,000 rpm for 15 min. The supernatant was then discarded, and the sample was resuspended in 500 μL of 75% ethanol and centrifuged at 4°C and 12,000 rpm for 15 min. Finally, the supernatant was discarded, and an appropriate amount of DEPC water was added to dissolve the precipitate. RNA samples were extracted from the brain tissue, reverse transcriptase was added, and the mixture was reverse-transcribed into 2000 ng of cDNA. RT-qPCR was then used to detect the viral DNA copy number (immediate early gene U) of compound JD13. L 54, Early gene U L 52, Late-stage gene U L 27) The role of expression. The primers and their corresponding sequences involved in the RT-qPCR experiment are shown below:

[0105] U L 54-F: 5'-GGCGGACATTAAGGACATTG-3' (SEQ ID NO.9);

[0106] U L54-R: 5'-TGGCCGTCAACTCGCAGA-3' (SEQ ID NO. 10);

[0107] U L 52-F: 5'-GACCGACGGGTGCGTTATT-3' (SEQ ID NO. 11);

[0108] U L 52-R: 5'-GAAGGAGTCGCCATTTAGCC-3' (SEQ ID NO. 12);

[0109] U L 27-F: 5'-GCCTTCTTCGCCTTTCGC-3' (SEQ ID NO. 13);

[0110] U L 27-R: 5'-CGCTCGTGCCCTTCTTCTT-3' (SEQ ID NO. 14).

[0111] Experimental results: Figure 11 The image shows the results of HSV-1 infection-induced Hsp90α protein expression in vivo. The results show that HSV-1 infection (herpesvirus encephalitis HSE model group) promotes the expression of Hsp90α in brain tissue. Figure 12 The compound JD13 was used to measure the symptoms of herpesvirus encephalitis and the HSV-1 gene copy number in brain tissue. L 54. U L 52. U L The results of the detection of expression 27 show that compound JD13 can significantly alleviate the symptoms of herpesvirus encephalitis and significantly inhibit viral gene expression in brain tissue induced by HSV-1 infection.

[0112] Example 8: Determination of the secretion level of virus-induced I-IFN by the target protein Hsp90α

[0113] The experimental method is as follows: Hsp90α was knocked down, and then microglia were infected with HSV-1 (MOI 1) for 12 h. After that, the secretion level of IFNβ in the culture supernatant and serum was detected by ELISA kit.

[0114] Experimental results: Figure 13 The image shows the results of HSV-1-induced INFβ secretion levels by knocking down Hsp90α. The results show that knocking down Hsp90α significantly promotes the secretion of IFNβ in the supernatant of HSV-1-infected microglia.

[0115] Example 9: In vitro drug binding to target protein Hsp90α affects the function of TRIM56.

[0116] Immunoprecipitation was used to determine the interaction between compound JD13 and TRIM56 and cGAS, as well as the ubiquitination level of cGAS, mediated by Hsp90α. The specific experimental method is as follows:

[0117] 1) Hsp90α was overexpressed, and then co-treated with HSV-1 (MOI 1) and compound JD13 (100 nM) for 12 h. The supernatant was discarded. After washing once with 3 mL of pre-cooled PBS, 300 μL of IP lysis buffer was added. Protein samples were scraped from the cells and lysed at 4 °C for 30 min. The protein concentration was determined by BCA. A portion of the supernatant (50 μL) was retained as the total cell lysis buffer. The remaining equal volume of sample was incubated overnight with the specified cGAS antibody (or an equal volume of IgG as a control) and protein A / G magnetic beads. The next day, the magnetic bead complex containing the sample and antibody was washed five times with PBS. 60 μL of 5× loading buffer was added and boiled to elute the protein from the magnetic beads. The protein was then used for subsequent immunoblotting analysis.

[0118] 2) After overexpressing Hsp90α, the cells were co-treated with HSV-1 and compound JD13 (100 nM) for 12 h, and the supernatant was discarded. After washing once with 3 mL of pre-cooled PBS, 300 μL of IP lysis buffer was added. Protein samples were scraped from the cells and lysed at 4 °C for 30 min. The protein concentration was determined by BCA. A portion of the supernatant (50 μL) was retained as the total cell lysis buffer. The remaining equal volume of sample was incubated overnight with the specified cGAS antibody (or an equal volume of IgG as a control) and protein A / G magnetic beads. The next day, the magnetic bead complex containing the sample and antibody was washed five times with PBS. Each group of magnetic bead complex samples was evenly divided into two aliquots. One aliquot was boiled with 60 μL of 5× loading buffer to elute the protein from the magnetic beads for Western blot analysis. The other aliquot was added with 20 μL of proteinase. Proteinase K was used to elute the protein from the magnetic beads, and the supernatant was transferred to a new EP tube for viral DNA extraction. Exogenous EGFP, 200 μL LBB5, and 200 μL PBS were added to a sample tube containing 20 μL Proteinase K, and the mixture was centrifuged and incubated at 56°C for 15 min. 250 μL of anhydrous ethanol was added, and the mixture was vortexed for 15 s and allowed to stand for 5 min. The solution was transferred to a centrifuge column, centrifuged at 12000 × g for 1 min, and the eluent was discarded. The sample was washed twice with WB5, centrifuged at 2000 × g for 1 min, and the eluent was discarded. The centrifuge column was transferred to a new EP tube, and 20 μL of DEPC water was added to elute the DNA. RT-qPCR was used to detect the viral DNA copy number. The primer sequences used in the RT-qPCR experiment included:

[0119] U L 27-F: 5'-GCCTTCTTCGCCTTTCGC-3' (SEQ ID NO. 13);

[0120] U L 27-R: 5'-CGCTCGTGCCCTTCTTCTT-3' (SEQ ID NO. 14);

[0121] U L 47-F: 5'-ACGATGATGATGAGGTTCCC-3' (SEQ ID NO. 15);

[0122] U L 47-R: 5'-ACGATGATGATGAGGTTCCC-3' (SEQ ID NO. 16).

[0123] Experimental results: Figure 14 This is a graph showing the verification results of the interaction between compound JD13 and cGAS and TRIM56 via Hsp90α. Figure 15 This figure shows the validation results of compound JD13's effect on the binding of cGAS to viral DNA via Hsp90α. The results indicate that after expressing Hsp90α, co-treatment with HSV-1 (MOI 1) and compound JD13 (100 nM) significantly reduced the interaction between TRIM56 and cGAS after Co-IP TRIM56. Simultaneously, RT-qPCR results after Co-IP cGAS showed that compound JD13 affected the binding ability of the TRIM56 interacting protein cGAS to viral DNA. Therefore, compound JD13, through Hsp90α, acts on the function of TRIM56 ubiquitinizing cGAS, activating cGAS and enhancing its binding to viral DNA, thus promoting I-IFN production.

[0124] Example 10: Verification of the effect of compound JD13 on promoting I-IFN expression via TRIM56

[0125] The expression of I-IFN mediated by compound JD13 via TRIM56 was determined using Western blotting. The specific experimental method is as follows: Microglial cells (BV2) were seeded at 350,000 cells / well in 6-well plates and cultured overnight in a 5% CO2 incubator at 37°C. The next day, the appropriate amount of siRNA-TRIM56 was diluted with the corresponding reagents (transfection buffer, serum-free DMEM), vortexed for 10 seconds, and centrifuged for 2 seconds. The appropriate amount of transfection reagent was added to the diluted siRNA mixture, vortexed for 10 seconds, and centrifuged for 2 seconds. Subsequently, the transfection complex was incubated at room temperature for 10 minutes (5 minutes for the siRNA mixture). After incubation, the transfection complex was added dropwise to the culture dish using a pipette tip while slowly mixing the liquid in the plate to ensure even distribution. After 6 hours of transfection, the medium was replaced with 10% FBS complete DMEM medium, and the cells were cultured for another 48 hours to obtain the TRIM56 knockdown cell line. Subsequently, cells were infected with HSV-1 (MOI 1) and co-treated with compound JD13 for 12 h. Protein samples were obtained by SDS lysis. Protein concentration was then determined using the BCA protein assay, specifically by SDS-PAGE electrophoresis of 40 μg of protein. The PVDF membrane was blocked with 5% skim milk at room temperature for 1 h, followed by overnight incubation at 4°C in the presence of a specific antibody. Later, Western blotting was used to determine the phosphorylation activation level of I-IFN upstream regulatory proteins regulated by compound JD13 via TRIM56.

[0126] Experimental results: Figure 16 The image shows the detection results of the activation of upstream I-IFN proteins by compound JD13 via TRIM56. The results show that siRNA silencing TRIM56 can significantly inhibit the phosphorylation of upstream I-IFN regulatory proteins TBK1 and IRF3 regulated by JD13, indicating that compound JD13 promotes I-IFN expression through TRIM56.

[0127] The results above indicate that the compound JD13 described in this invention can enhance the body's antiviral innate immunity by targeting Hsp90α, activating and enhancing TRIM56 ubiquitination, and quantitatively regulating the increase of I-IFN expression, thereby treating infectious encephalitis. It also shows therapeutic efficacy against infectious encephalitis resistant to nucleoside analogues such as acyclovir. The evaluation method for the treatment of infectious encephalitis with compound JD13 can be achieved by calculating and predicting the binding of compound JD13 to the target protein Hsp90α, thus providing a preliminary evaluation of the antiviral activity and site of compound JD13. Furthermore, the efficacy of the drug in treating infectious encephalitis can be evaluated in vitro and in vivo by assessing the binding of compound JD13 to the Hsp90α target protein after infection, the TRIM56 ubiquitination status, and the I-IFN expression. The calculated prediction results are consistent with the in vitro drug efficacy evaluation results and are unified with the in vivo pharmacodynamic results. Both the calculated prediction and in vitro drug evaluation methods provide a basis for drug screening in infectious encephalitis.

[0128] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis, wherein the Hsp90α-targeting compound has the structure shown in Formula 1: In Formula 1, R is selected from amino heterocyclic groups.

2. The application of a Hsp90α-targeting compound in the preparation of a therapeutic drug for infectious encephalitis caused by nucleoside analogue-resistant viruses, wherein the Hsp90α-targeting compound has the structure shown in Formula 1: In Formula 1, R is selected from amino heterocyclic groups.

3. The application according to claim 1 or 2, characterized in that, The target Hsp90α compound has the structure shown in Formula 1-1, Formula 1-2, or Formula 1-3:

4. The application according to claim 3, characterized in that, The Hsp90α-targeting compound binds to the Hsp90α protein, and the binding site between the Hsp90α-targeting compound and the Hsp90α protein is LYS58.

5. The application according to claim 1 or 2, characterized in that, The infectious encephalitis mentioned is herpesvirus encephalitis.

6. The application according to claim 5, characterized in that, The herpesvirus encephalitis mentioned is herpesvirus encephalitis caused by HSV-1.

7. The application according to claim 1 or 2, characterized in that, The infectious encephalitis treatment drug activates the TRIM56-cGAS-STING immune pathway in the organism through the Hsp90α-targeting compound, promotes I-IFN production, and enhances the body's immune function to treat herpesvirus encephalitis caused by HSV-1.

8. The application according to claim 2, characterized in that, The nucleoside analogues include acyclovir.

9. The application according to claim 1 or 2, characterized in that, The dosage forms of the drugs for treating infectious encephalitis include oral preparations, topical preparations, or injectable preparations.

10. The application according to claim 9, characterized in that, The oral preparations include tablets, capsules, pills, sugar-coated preparations, sustained-release preparations, controlled-release preparations, or suspensions; the topical preparations include lotions, creams, gels, ointments, microneedles, sprays, or drops; and the injectable preparations include injection solutions or powder injections.

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