Use of YAP pathway activator in preparation of Anti-hepatotropic virus medicament

Abstract

A use of a YAP pathway activator in the preparation of an anti-hepatotropic virus medicament. In in vivo and in vitro hepatotropic virus replication models, it has been found that using the YAP pathway activator can significantly inhibit the replication of hepatotropic viruses such as hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus and the expression of viral antigens. The antiviral effect of the activator is mainly achieved by means of regulation of the YAP signaling pathway and the fact that inhibiting YAP can restore viral replication. The described YAP pathway activator may be specifically a LATS1 / 2 kinase inhibitor, such as TDI-011536 or TRULI. The YAP pathway activator has no significant cytotoxicity and has a good antiviral effect.

Description

Application of YAP pathway activators in the preparation of anti-hepatitis virus drugs

[0001] This application claims priority to Chinese patent application 202410660624X, filed on 2024 / 05 / 27. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical technology, specifically to the application of Hippo-YAP pathway inhibitors in the treatment of hepatotropic viruses, and more specifically to the application of YAP pathway activators in the preparation of anti-hepatotropic virus drugs. Background Technology

[0003] The liver, as the largest solid organ in the human body, possesses vital biological functions and unique characteristics. Hepatotropic viruses rely on liver-specific enrichment factors to complete their life cycle. Their infection and replication within the liver can cause acute and chronic hepatitis, and further lead to cirrhosis and liver cancer, seriously endangering health. Among these, hepatitis B virus (HBV) is a major cause of viral hepatitis, and chronic infection can lead to numerous liver diseases, including chronic hepatitis B, cirrhosis, and liver cancer. However, current treatments, including antiviral drugs targeting the reverse transcription process and interferon, can control but not effectively cure chronic hepatitis B. Therefore, developing antiviral methods with novel mechanisms is of great significance. Furthermore, existing antiviral drugs and interferon treatments suffer from limited antiviral efficacy, high adverse reactions, and the inability to effectively cure chronic infectious diseases.

[0004] The Hippo-YAP signaling pathway is a crucial signaling network regulating organ size, cell growth, and tissue regeneration. This pathway controls cell proliferation, death, and differentiation through the activity of a series of kinases and transcription factors. Key components include MST1 / 2 (mammalian Ste20-like kinase) and LATS1 / 2 (large tumor suppressor protein kinase), which regulate the downstream transcriptional coactivator YAP (Yes-associated protein) via phosphorylation. In the normal, inactive state, YAP and Taz are phosphorylated and retained in the cytoplasm, preventing them from entering the nucleus to exert their effects. When the Hippo pathway is inhibited, unphosphorylated YAP can enter the nucleus, activating the expression of various growth factors and genes promoting cell proliferation and survival. This mechanism plays a critical role in the size control and regeneration of organs such as the liver. For example, after liver damage, inhibition of the Hippo pathway can promote hepatocyte proliferation, thereby rapidly restoring liver function.

[0005] However, there are currently no studies or reports on achieving anti-hepatotropic virus effects by regulating the YAP pathway. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of YAP pathway activators in the preparation of anti-hepatitis virus drugs, thereby solving problems such as limited antiviral efficacy, high adverse reactions, and inability to effectively cure chronic infectious diseases in related technologies.

[0007] In their prior research, the inventors discovered that in in vitro and in vivo hepatitis B virus replication models, high hepatocyte basal stiffness and activation of related mechanosensing pathways significantly inhibited viral replication. Exogenous use of key mechanosensing-related YAP pathway kinase regulators, such as small molecule compounds like XMU-MP-1, GA-017, and TRULI, significantly inhibited viral replication and viral antigen expression. The antiviral effect of these key mechanosensing-related YAP pathway kinase regulators is mainly achieved by downregulating hepatocyte-enriched transcription factors dependent on hepatotropic virus replication and upregulating a series of molecules with antiviral effects (see CN202310426492X - Application of Mechanosensing-Related YAP Pathway Regulators in the Preparation of Anti-Hepatotropic Virus Drugs). Through further screening and research, the inventors found that the YAP pathway activator TDI-011536 used in this invention exhibits even better anti-hepatotropic virus effects in vitro and in vivo.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides the application of YAP pathway activators in the preparation of anti-hepatoviral drugs, wherein the YAP pathway activators include LATS1 / 2 kinase inhibitors, and the LATS1 / 2 kinase inhibitors include at least one of TDI-011536 and TRULI.

[0010] The chemical structural formula of TDI-011536 is shown below:

[0011] The chemical structural formula of the above TRULI is shown below:

[0012] Furthermore, the YAP pathway activator is TDI-011536. TDI-011536 effectively activates YAP protein in the liver without significant hepatotoxicity.

[0013] Furthermore, the hepatotropic viruses include hepatitis A virus (single-stranded positive-sense RNA virus), hepatitis B virus (DNA virus), hepatitis C virus (single-stranded positive-sense RNA virus), hepatitis D virus (single-stranded negative-sense RNA virus), hepatitis E virus (single-stranded positive-sense RNA virus), and other liver-centric viruses.

[0014] Furthermore, the anti-hepatotropic virus drug includes drugs that combat hepatotropic virus infections such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E; preferably, the anti-hepatotropic virus drug is a drug that combats hepattropic virus infections such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E.

[0015] Furthermore, the YAP pathway activator is used to prepare drugs that inhibit hepatotropic virus replication and viral antigen expression.

[0016] Furthermore, the YAP pathway activator is used to prepare drugs that inhibit YAP-reversible viral replication.

[0017] Specifically, TDI-011536 has a good inhibitory effect on hepatitis B virus (HBV) RNA and hepatitis B surface antigen HBsAg, and its effect mainly occurs in the rcccDNA transcription and translation stages; both TDI-011536 and TRULI inhibit the replication of hepatitis C virus (HCV) and can effectively reduce HCV RNA levels; both TDI-011536 and TRULI have a significant inhibitory effect on hepatitis D virus (HDV) antigen HDAg; in addition, TDI-011536 also has a significant inhibitory effect on hepatitis A virus (HAV) and hepatitis E virus (HEV) RNA.

[0018] Furthermore, the YAP pathway activator regulates the Hippo-YAP signaling pathway, thereby activating YAP by inhibiting upstream molecules of YAP.

[0019] Furthermore, the preparation of the anti-hepatitis virus drug also includes the use of a pharmaceutically acceptable carrier. Such carriers may be diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings conventionally used in the art.

[0020] Furthermore, the preparation of the anti-hepatotropic virus drug also includes the use of other anti-hepatotropic virus active ingredients, such as interferon, to achieve the combined administration of different drugs.

[0021] Furthermore, the dosage forms of the anti-hepatitis virus drugs include solid dosage forms, liquid dosage forms, and semi-solid dosage forms, specifically including tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, drop pills, dry extracts, injections, or infusions.

[0022] Furthermore, the drug can be administered via conventional methods in the art, including but not limited to injection or oral administration. Injection administration includes routes such as intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0023] Furthermore, in the preparation of anti-hepatitis virus drugs, the YAP pathway activator is used as the active ingredient, and its content can be 0.1wt% to 99.9wt%, such as 1wt% to 80wt%, 5wt% to 50wt%, 10wt% to 20wt%, etc.

[0024] Furthermore, the standard dosage of the anti-hepatotropic virus drug is: YAP pathway activator 0.1 mg / day to 1000 mg / day. For example, mice are treated with intraperitoneal injection of 30 to 50 mg / kg / day for a period of 5 to 15 days (specifically, mice can be treated with intraperitoneal injection of 40 mg / kg / day for a period of 8 days).

[0025] The present invention also provides a pharmaceutical composition for treating hepatotropic viruses, comprising TDI-011536 and TRULI, and a pharmaceutically acceptable carrier.

[0026] Preferably, in the anti-hepatitis virus drug composition, the mass ratio of TDI-011536 to TRULI can be adjusted according to actual needs. For example, the mass ratio of TDI-011536 to TRULI is 1:1, 1:2, 1:3, 1:4 or 1:5, or the mass ratio of TRULI to TDI-011536 is 1:1, 1:2, 1:3, 1:4 or 1:5.

[0027] Preferably, the anti-hepatophilic drug composition is a drug composition that is effective against one or more of the following: hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.

[0028] The present invention also provides a method for treating diseases caused by hepatotropic viruses, comprising administering to a subject the YAP pathway activator described above or the drug combination described above.

[0029] Preferably, the method of administration can be conventional in the art, including but not limited to injection or oral administration.

[0030] Preferably, the dosage of the YAP pathway activator or the pharmaceutical composition during administration can be conventional in the art, such as 0.1 mg / day to 1000 mg / day.

[0031] For example, mice were treated with intraperitoneal injection of 30–50 mg / kg / day for 5–15 days (specifically, mice were treated with intraperitoneal injection of 40 mg / kg / day for 8 days).

[0032] The present invention also provides a method for inhibiting hepatotropic virus replication, inhibiting hepatotropic virus antigen expression, inhibiting YAP-reversible viral replication, or inhibiting hepatotropic virus RNA levels, comprising administering to a subject the YAP pathway activator described above or the drug combination described above.

[0033] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0034] This invention is the first to discover that, in in vitro and in vivo hepatotropic virus replication models, the use of Hippo-YAP pathway inhibitors, through the inhibition of upstream molecules of YAP, can activate YAP and significantly inhibit the replication and viral antigen expression of hepatotropic viruses such as hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis C virus, and hepatitis E virus. The antiviral effect is mainly achieved by regulating the Hippo-YAP signaling pathway; inhibiting YAP can restore viral replication. Simultaneously, the LATS1 / 2 kinase inhibitor, specifically TDI-011536, as a YAP pathway activator, exhibits no significant cytotoxicity and good antiviral efficacy. These results suggest the application of Hippo-YAP pathway inhibitors (specifically YAP pathway activators) in the preparation of anti-hepatotropic virus drugs. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0036] Figure 1 is a schematic diagram showing the results of activating YAP protein in the liver by applying YAP activator (TDI-011536) in one embodiment of the present invention;

[0037] Figure 2 is a schematic diagram of the anti-hepatitis B virus effect of applying YAP activator (TDI-011536) in one embodiment of the present invention;

[0038] Figure 3 is a schematic diagram showing the effect of applying YAP activator (TRULI, TDI-011536) and inhibitor (Lat.A) on hepatitis C virus infection in one embodiment of the present invention.

[0039] Figure 4 is a schematic diagram showing the effect of applying YAP pathway activator (TRULI, TDI-011536) and inhibitor (Lat.A) on hepatitis D virus infection in one embodiment of the present invention.

[0040] Figure 5 is a schematic diagram showing the effect of applying YAP pathway activator (TDI-011536) on hepatitis A virus infection in one embodiment of the present invention.

[0041] Figure 6 is a schematic diagram showing the effect of applying YAP pathway activator (TDI-011536) on hepatitis E virus infection in one embodiment of the present invention. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0043] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0044] The reagents used in the following examples were: TDI-011536 and TRULI purchased from Selleck; Lat.A purchased from Santa Cruz; C57BL / 6 mice purchased from Jicui Yaokang; HBV replication plasmid and Huh7.5 cells were self-owned and routinely stored in Dulbecco modified medium supplemented with 10% fetal bovine serum (Biological Industries), 25mM HEPES (Gibco), and Non-Essential. HAV virus particles, HCV virus particles, HDV virus particles, and HEV virus particles were all purified using conventional preparation methods. HepG2 NTCP Both the cells and Huh7.5.1 cells are conventional cell lines.

[0045] The detection method used in the following embodiments:

[0046] (1) Western blot assay (immunoblotting): Protein samples were collected using RIPA lysis buffer containing protease inhibitors, 5× loading buffer was added, and the mixture was heated at 100°C for 5 min, followed by incubation on ice for 5 min. Proteins were separated using a 10% SDS-PAGE gel (protein loading volume: 10 μL) and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h, the PVDF membrane was cut, and incubated overnight with the corresponding antibodies. The PVDF membrane incubated with the primary antibody was washed with TBST, the corresponding secondary antibody was added and incubated for 2 h, and then the PVDF membrane incubated with the secondary antibody was washed with TBST. The membrane was then subjected to Western blotting. TM The chemiluminescent reagent was applied for 2 minutes, then the sample was placed in a darkroom and exposed with photographic film. After the film was completely dry, the results were analyzed and photographed.

[0047] (2) ELISA detection: Enzyme-linked immunosorbent assay, mouse serum was diluted 10 times to detect HBeAg and diluted 100 times to detect HBsAg.

[0048] (3) Northern Blot detection: Similar to Western Blot, the difference is that NB detects RNA levels; WB detects protein levels.

[0049] (4) Use the Hirt extraction method to detect rcccDNA. For specific steps, please refer to https: / / jmi.fudan.edu.cn / fileup / HTML / 11868.htm.

[0050] (5) Luciferase reporter assay: Gluc substrate was added to the cell culture supernatant and the fluorescence level was detected.

[0051] (6) RT-qPCR experiment: Cells were lysed in Trizol reagent (Invitrogen), RNA was purified from the Trizol extract, and then processed using PrimeScript according to TaKaRa's gDNA Eraser (Perfect Real-Time). TM The RTreagent Kit is used for reverse transcription. Real-time PCR is performed on the cDNA sample using the specific primer sequences listed. Premix Ex Taq TM Tli RNaseH Plus) was used to calculate the relative RNA level using the 2^(-△△Ct) method, with GAPDH as the internal control gene.

[0052] The relevant primer sequences are as follows:

[0053] GAPDH: F 5'-GGTATCGTGGAAGGACTCATGA-3' (SEQ ID NO: 1);

[0054] R 5'-ATGCCAGTGGCTTCCCGTTCAGC-3' (SEQ ID NO: 2);

[0055] HCV: F 5'-CCCTGTGAGGAACTACTGTCTTCACGC-3' (SEQ ID NO: 3);

[0056] R 5'-GCTCATGGTGCACGGTCTACGAGAC-3' (SEQ ID NO: 4);

[0057] HAV-VP1: F 5'-GGTCTTGCCGTTGATACTCCTTG-3' (SEQ ID NO: 5);

[0058] R 5'-TCCCTGTTCTCCTTGTGTTAAATCTG-3' (SEQ ID NO: 6);

[0059] Capsid-HEV: F 5'-CCGATGTCCGTATCCTTGTTCAAC-3' (SEQ ID NO: 7);

[0060] R 5'-TCAGCAACGCCAGAGGTCTC-3' (SEQ ID NO:8).

[0061] Example 1 - TDI-011536 effectively activates YAP protein in the liver.

[0062] This embodiment verifies the activation effect of TDI-011536 on YAP protein. The specific operation steps include:

[0063] Ten C57BL / 6 mice were administered TDI-011536 intraperitoneally at a dose of 40 mg / kg. The livers of the mice were collected at 0, 2, 4, 8, and 24 hours (two mice at each time point). Western blotting was used to detect the protein levels of YAP (total YAP), pYAP (inactivated form of YAP), and Active YAP (activated form of YAP).

[0064] The experimental results are shown in Figure 1. The results show that TDI-011536 can effectively activate YAP protein in the liver after administration, and it can be used as a YAP pathway activator.

[0065] Example 2 - Anti-hepatitis B virus effect of YAP pathway activator

[0066] This embodiment verifies the anti-hepatitis B virus effect of the YAP pathway activator TDI-011536 in a mouse HBV replication model, specifically including the following steps:

[0067] (1) Experimental procedures;

[0068] As shown in part a of Figure 2, ten C57BL / 6 mice were selected. First, HBV replication plasmids were injected via high-pressure hydrodynamic tail vein injection: 4 μg / mouse of prccc plasmid and 4 μg / mouse of pCMV-Cre plasmid, prepared as PBS solution at 10% of the mouse's body weight. This was injected via tail vein within 5–8 seconds to induce HBV expression (modeling). One day after plasmid injection, the mice were divided into a Mock group (DMSO, 5 mice per group) and a treatment group (TDI-011536, 5 mice per group). TDI-011536 was administered intraperitoneally at 40 mg / kg daily for 8 days (treatment). The control group received the same amount of DMSO. During the treatment period, serum viral antigen indicators (HBsAg, HBeAg, ALT / AST / ALB) were measured every two days via orbital blood sampling. Eight days after treatment, the mice were sacrificed, and their livers were harvested to detect other tissue damage and virological indicators (H&E, IHC, HBV RNA, rcccDNA).

[0069] (2) Experimental results;

[0070] 1) The serum HBV antigen level was detected by ELISA, and the experimental results are shown in part b of Figure 2. On day 8, the serum HBsAg level in the treatment group (TDI-011536) was significantly lower than that in the Mock group (DMSO), with an average decrease of 87.2%, but there was no significant difference in HBeAg.

[0071] 2) Northern blotting was used to detect HBV RNA levels in tissues, and the results are shown in part c of Figure 2. On day 8, the HBV RNA level in the liver of the drug-treated group (TDI-011536) was significantly lower than that of the Mock group (DMSO).

[0072] 3) The H&E staining results are shown in part d of Figure 2, indicating that the liver tissue sections of the treatment group (TDI-011536) and the Mock group (DMSO) have similar morphology. HBsAg histochemical staining results showed that the number of HBsAg antigen-positive cells in the liver was significantly lower in the treatment group (TDI-011536) compared to the Mock group (DMSO).

[0073] 4) The detection results of rcccDNA are shown in part e of Figure 2. rcccDNA, short for recombinant cccDNA, is an HBV transcription template similar to HBV cccDNA formed by Cre enzyme cleaving prccc after tail vein injection (mixed injection of prccc and Cre plasmids). It can transcribe HBV RNA and then translate it to produce HBV antigens (HBsAg, HBeAg). The results showed no significant difference between the drug-treated group (TDI-011536) and the Mock group, indicating that the effect of the drug-treated group mainly occurred in the transcription and translation of rcccDNA.

[0074] 5) ALB is serum albumin, an important marker of normal liver function. The detection results are shown in part f of Figure 2. The results showed no significant difference between the drug-treated group (TDI-011536) and the Mock group, indicating that there was no hepatotoxicity at the working concentration of the drug-treated group.

[0075] 6) ALT / AST is an important indicator of liver injury, and its detection results are shown in part g of Figure 2. The results showed that there was no significant difference between the drug-treated group (TDI-011536) and the Mock group, which also indicates that there is no hepatotoxicity at the working concentration of the drug-treated group.

[0076] Example 3 - Anti-hepatitis C virus effect of YAP pathway activator

[0077] This embodiment verifies the anti-hepatitis C virus efficacy of YAP pathway activators (TRULI, TDI-011536) based on the Huh7 cell line. Specific operational steps include:

[0078] (1) Huh7.5 cells were induced at a rate of 0.2 × 10⁻⁶. 6 Cells / mL, 0.3 mL / well, 48-well plates were seeded; the effects of three drugs (TRULI 10 μM, TDI-011536 3 μM, Lat.A 0.1 μg / mL) on YAP were detected in Huh7.5 cells. Western blot results are shown in part a of Figure 3, which show that the YAP pathway activators TRULI and TDI-011536 can significantly reduce the protein level of pYAP (YAP inactivated state), while the YAP pathway inhibitor Lat.A can increase the level of pYAP.

[0079] (2) Huh7.5 cells were cultured at a rate of 0.2 × 10⁻⁶. 6Cells / mL, 0.3 mL / well, 48-well plate seeding; Huh7.5 cells were infected with HCV (HCV virus particles were added to the well plate at MOI=2, the infection process lasted for 1 day, and the medium was changed afterward), and treated with drugs (the drug stock solution was diluted to the specified concentration, and the medium was changed every 2 days; the concentrations of each drug were: TRULI 10 μM, TDI-0115363 μM, Lat.A 0.1 μg / mL, IFNα 21 ng / mL). The experimental procedure is shown in part b of Figure 3. Samples were collected on Day 4, and the fluorescence intensity in the supernatant and the intracellular HCV RNA level were detected (for specific methods, see: Xiang C et al. Long-term functional maintenance of primary human hepatocytes in vitro. Science. 2019 Apr 26; 364(6438):399-402. Supplementary Fig. S27 / Supplementary HCV infection and Detection of HCV product).

[0080] Experimental results:

[0081] 1) Fluorescence reporter assay: The HCV used in this example was modified to secrete luciferase into the cell supernatant during replication. The fluorescence intensity in the supernatant reflects the degree of HCV replication activity. To quantify luciferase activity, supernatant was taken from each well and mixed with an equal volume of 2×passive lysis buffer (Promega). Luciferase activity was measured using Renilla luciferase substrate (Promega) according to the manufacturer's method. The results are shown in part c of Figure 3. The results showed that YAP pathway activators had an inhibitory effect, while YAP pathway inhibitors had a promoting effect. Additionally, IFNα2 served as a positive control; compared to the Mock group, IFNα2 strongly inhibited HCV replication.

[0082] 2) The intracellular HCV RNA level at the experimental endpoint (Day 4) was detected using RT-qPCR, and the results are shown in part d of Figure 3. The results showed that the inhibition rates of HCV RNA were as follows: TRULI (10 μM) (68.5% decrease), TDI-011536 (3 μM) (65.5% decrease), Lat.A (0.1 μg / ml) (110% increase), and IFNα2 (1 ng / ml) (96.8% decrease).

[0083] As can be seen from the above embodiments, the present invention has found in in vitro and in vivo hepatotropic virus replication models that by using a YAP pathway activator (specifically TDI-011536), the replication of hepatotropic viruses such as hepatitis B virus and hepatitis C virus and viral antigen expression can be significantly inhibited. Its antiviral effect is mainly achieved by regulating the YAP signaling pathway. Inhibiting YAP can restore viral replication. The above-mentioned YAP pathway activator TDI-011536 has no obvious cytotoxicity and has good antiviral effect.

[0084] Example 4 - Efficacy of YAP Pathway Activator against Hepatitis D Virus (HDV)

[0085] This embodiment is based on HepG2. NTCP The cell lines validated the anti-hepatitis D virus efficacy of the YAP pathway activators (TRULI, TDI-011536). Specific procedures included:

[0086] HepG2 NTCP Cells were grown at a rate of 0.4 × 10⁻⁶. 6 Cells were seeded in 48-well plates at 0.3 mL / well. Cells were infected with HDV (HDV virus particles were added to the plates at 0.5 IU / cell; the culture medium was supplemented with 4% PEG8000 and 2.5% DMSO for one day, with the medium changed the following day), and treated with drugs (drug stock solutions were diluted to specified concentrations, and the medium was changed every 2.5 days; the concentrations of each drug were: TRULI 10 μM, TDI-01 15363 μM, and Lat. A 0.1 μg / mL). The experimental procedure is shown in Figure 4. Samples were collected on Day 5, and the intracellular HDAg expression ratio was detected by immunofluorescence staining. (For specific methods, see: Zhenfeng Zhang et al. Hepatitis D virus-induced interferon response and administered interferons control cell division-mediated virus spread. J. Hepatol. 2022, 77, 957-966. Supplementary materials and methods: HBV and HDV infection.)

[0087] Experimental results:

[0088] Immunofluorescence staining experiments. Using HDAg (Hepatitis DAntigen) primary antibody staining (as shown in Figure 4), on day 5 post-HDV infection, the intracellular HDAg positivity rate was significantly lower in the YAP activating group compared to the positive control (DMSO) group: approximately a 26-fold decrease in the TDI-011536 group (positive control / TDI-011536 = 4.96% / 0.19%), and approximately an 83-fold decrease in the TRULI group (positive control / TRULI = 4.96% / 0.06%).

[0089] As can be seen from the above embodiments, the present invention found in in vitro and in vivo hepatotropic virus infection models that the expression of hepatitis D virus antigen can be significantly inhibited by using YAP pathway activators (specifically TRULI and TDI-011536). The antiviral effect is mainly achieved by regulating the YAP signaling pathway and inhibiting YAP to restore viral replication. The above-mentioned YAP pathway activators have good anti-HDV effects.

[0090] Example 5 – Anti-hepatitis A virus (HAV) effect of YAP pathway activator

[0091] This embodiment verifies the anti-hepatitis A virus effect of the YAP pathway activator (TDI-011536) based on the Huh7.5.1 cell line. The specific operation steps include:

[0092] Huh7.5.1 cells were cultured at a rate of 0.4 × 10⁻⁶. 6 Cells were seeded at 0.5 mL / well in 24-well plates. Cells were infected with HAV (HAV virus particles were added to the plates at MOI=1, infection lasted 4 hours, and the medium was changed on the same day), and treated with a drug (the drug stock solution was diluted to the specified concentration, and the medium was changed every 2 days; the drug concentration was TDI-0115363 μM). The experimental procedure is shown in Figure 5. Samples were collected on Day 4, and the relative content of HAV RNA in the cells was detected by RT-qPCR. (For specific methods, see: Wang Jiang et al. Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles. J Extracell Vesicles 2020 Vol.9 Issue 1 Pages 1716513. Figure 6.)

[0093] Experimental results:

[0094] RT-qPCR: Using the HAVVP1 primer sequence (see above) and GAPDH as an internal control, the relative HAV RNA content in the YAP-activated group (+HAV_TDI-011536) compared to the untreated group (+HAV) was detected. On day 4 after HAV infection, the intracellular HAV content in the YAP-activated group (+HAV_TDI-011536) was significantly lower than that in the untreated group (+HAV): the HAV RNA content decreased by 73.8% (as shown in Figure 5).

[0095] As can be seen from the above embodiments, the present invention found in in vitro and in vivo hepatophilic virus infection models that the use of YAP pathway activator (specifically TDI-011536) can significantly inhibit the intracellular hepatitis A virus RNA content. Its antiviral effect is mainly achieved by regulating YAP activity. The above-mentioned YAP pathway activator TDI-011536 has no obvious cytotoxicity and has good antiviral effect.

[0096] Example 6 – Anti-hepatitis E virus (HEV) effect of YAP pathway activator

[0097] This embodiment verifies the anti-hepatitis E virus efficacy of the YAP pathway activator (TDI-011536) based on the Huh7.5.1 cell line. The specific operation steps include:

[0098] Huh7.5.1 cells were cultured at a rate of 0.4 × 10⁻⁶. 6 Cells were seeded at 0.5 mL / well in 24-well plates. Cells were infected with HEV (HEV virus particles were added to the plates at an MOI of 0.1, and the infection process lasted 4 hours, with medium changed on the same day), and treated with drugs (the drug stock solution was diluted to the specified concentration, and the medium was changed every 2 days; the drug concentration was: TDI-01 15363 μM, IFNα 2 10 ng / mL). The experimental procedure is shown in Figure 5. Samples were collected on Day 4, and the relative content of intracellular HEV RNA was detected by RT-qPCR. (For specific methods, see: Qiang Ding et al. Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription.mBio 2018 Vol.9 Issue 3. Figure 6.)

[0099] Experimental results:

[0100] RT-qPCR: Using the Capsid-HEV primer sequence (see above) and GAPDH as an internal control, the relative HEV RNA content in the YAP-activated group (+HEV_TDI-011536) compared to the untreated group (+HEV) was detected. On day 4 post-HEV infection, the intracellular HEV content in the YAP-activated group (+HEV_TDI-011536) was significantly lower than that in the untreated group (+HEV): HEV RNA content decreased by 49.4%. As a positive control, the IFN-treated group (+HEV_IFNα2 10 ng / ml) showed a 65.2% decrease in HEV RNA content (as shown in Figure 6).

[0101] As can be seen from the above embodiments, the present invention found in in vitro and in vivo hepatitis E virus infection models that the use of YAP pathway activator (specifically TDI-011536) can significantly inhibit the intracellular hepatitis E virus RNA content. Its antiviral effect is mainly achieved by regulating YAP activity. The above-mentioned YAP pathway activator TDI-011536 has no obvious cytotoxicity and has good antiviral effect.

[0102] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of YAP pathway activators in the preparation of anti-hepatotropic virus drugs, characterized in that, The YAP pathway activator includes a LATS1 / 2 kinase inhibitor, which includes at least one of TDI-011536 and TRULI.

2. The application of the YAP pathway activator according to claim 1 in the preparation of anti-hepatotropic virus drugs, characterized in that, The YAP pathway activator is TDI-011536; and / or, the hepatotropic virus includes one or more of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.

3. The application according to any one of claims 1 to 2, characterized in that, The YAP pathway activator is used to prepare drugs that inhibit hepatotropic virus replication and viral antigen expression.

4. The application according to any one of claims 1 to 2, characterized in that, The YAP pathway activator is used to prepare drugs that inhibit YAP-reversible viral replication.

5. The application according to any one of claims 1 to 2, characterized in that, The YAP pathway activator regulates the Hippo-YAP signaling pathway, thereby activating YAP by inhibiting upstream molecules of YAP.

6. The application according to any one of claims 1 to 2, characterized in that, The preparation of the anti-hepatoviral drug also includes the use of a pharmaceutically acceptable carrier.

7. The application according to any one of claims 1 to 2, characterized in that, The dosage forms of the anti-hepatitis virus drugs include solid dosage forms, liquid dosage forms, and semi-solid dosage forms.

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