CN103063840A - Application of cellular target liver X receptor in preparation of drugs treating hepatitis C virus - Google Patents

Abstract

The invention discloses application of a cellular target liver X receptor in preparation of drugs treating hepatitis C virus (HCV), specifically a cellular target that can be used for screening anti-HCV drugs. The cellular target is a liver X receptor (LXR). The agonist GW3965 and T0901317 of the liver X receptor have significant anti-hepatitis C activity in invitro anti-hepatitis C virus experiments. The cellular target liver X receptor can be used for screening drugs treating the hepatitis C virus. Therefore, the cellular target LXR involved in the invention can be applied to drug development as a new anti-hepatitis C virus target, thus providing a new approach and means for treatment and cure of hepatitis C.

Description

Application of a cell target liver X receptor in the preparation of drugs for treating hepatitis C virus

technical field

The invention belongs to the technical field of medicine, and more specifically relates to the application of a cell target liver X receptor in the preparation of medicines for treating or preventing hepatitis C virus.

Background technique

Hepatitis C Virus (HCV), discovered in 1989, is the main pathogen causing non-A, non-B hepatitis after blood transfusion. About 170 million people worldwide are currently infected with HCV. HCV infection is distributed worldwide, the infection rate is about 3%, and 3 to 4 million new cases are added every year. According to the serological epidemiological survey data in my country, the positive rate of HCV in the general population is 3.2%. The main routes of transmission of HCV include blood transfusion or blood products, intravenous drug use, sexual contact, and mother-to-child transmission. HCV infection can easily lead to chronicity, only about 20% of HCV-infected patients can heal spontaneously, and about 80% of acutely infected patients will develop into chronic infection, and 20% of chronically infected patients will end up in the following 5 to 10 years It develops into cirrhosis and even liver cancer. In addition to affecting the liver, HCV can also cause lesions in other tissues and organs. Examples: mixed cryoglobulinemia, non-Hodgkin's lymphoma, and membranous proliferative glomerulonephritis.

Hepatitis C virus belongs to the genus Hepacivirinae of the Flaviviridae family in taxonomy. It is an enveloped single-stranded positive-sense RNA virus with a genome length of 9.4-9.6Kb. (5′nontranslated region, 5′NTR), a long open reading frame (openreading frame, ORF), and a 3′ nontranslated region (3′nontranslated region, 3′NTR). The ORF of HCV encodes a polyprotein precursor of about 3010-3040 amino acids, which is cleaved by the host cell protease and the protease encoded by the virus itself to produce at least 10 protein products. They are the structural proteins C, E1, E2, and P7 of the virus, which mainly constitute the capsid and envelope of HCV; the non-structural proteins of the virus, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, are involved in the processing and maturation of viral proteins and genome replication play a key role in. In addition, a frameshift reading frame was found in the Core coding region, and the protein encoded by it is called F protein, and its function is still unclear. The Eleventh International Symposium on HCV and Related Viruses (2004) developed and adopted a unified HCV classification standard based on the previous ones. According to this standard, HCV is divided into 6 major genotypes (1, 2, 3, 4, 5, 6) and several genotypes. There are significant regional differences in the distribution of HCV genotypes: in the United States and Western Europe, mainly types 1a and 1b; in Egypt in the Middle East, mainly type 4; in South Africa, mainly type 5; in Central Africa, mainly types 1 and 4; Mainly types 3 and 6. In my country, HCV 1b and 2a genotypes are more common, of which 1b is the main type.

The entire life cycle of HCV is completed in the cytoplasm, including the following major steps: (1) Adsorption and invasion: the combination of viral surface proteins and cell receptors determines the tropism and host specificity of virions. HCV currently only infects humans and chimpanzees. Human hepatocytes are the main infected cells, and other cells that have been reported to be infected include B lymphocytes and dendritic cells. The process of adsorption and invasion is completed by the binding of HCV envelope proteins E1 and E2 to HCV receptor molecules. These successively discovered receptors include: CD81, low-density lipoprotein receptor (LDLR), scavenger receptor SR-BI , Claudin-1, occludin, etc., there may be other new receptors. (2) Release of viral genome: HCV virions are internalized into cells by clathrin-mediated endocytosis, and released nucleocapsid and genome under the induction of low pH value. (3) Synthesis and processing of HCV polyprotein: the plus-strand genome of HCV is translated into a large polyprotein under the mediation of the 5′NTR ribosome entry site (IRES), and the polyprotein precursor is absorbed by the virus and the host Protease cleavage produces structural and nonstructural proteins. (4) Replication of the viral genome: HCV first synthesizes the negative strand using the positive strand RNA as a template, and then synthesizes the positive strand using the negative strand as a template. Studies have shown that HCV replication occurs in the membrane reticulum structure on the ER (endoplasmic reticulum). (5) Assembly of virus particles: The assembly of viral nucleocapsids usually includes the multimerization of capsid proteins and the packaging process of genome RNA by nucleocapsids. Recent studies have shown that the assembly of HCV virions may be lipid droplets in cells Finished around. (6) Maturation and release of virions: HCV virions may bud on the endoplasmic reticulum, and then release mature virions to the outside of the cell membrane through the Golgi apparatus.

At present, anti-HCV treatment methods are mainly limited to the combination therapy of polyethylene glycol-conjugated long-acting alpha interferon (peg-IFN) and ribavirin. The cure rate of this therapy for HCV is only 40% (type 1 HCV infection) to 80% (type 2 HCV infection), and the course of treatment is long, expensive, and side effects are obvious. Of course, with the deepening of research on HCV, many drugs targeting HCV protein are emerging. Telaprevir and Boceprevir belong to protease inhibitors (PI) in direct antiviral drugs. With the launch of these two direct antiviral drugs Telaprevir and Boceprevir in 2011, the standard treatment plan for hepatitis C will be significantly improved. However, there is currently no effective vaccine to prevent HCV infection. Therefore, HCV infection is still an important public health problem endangering the health of the global population.

Liver X receptor (liver X receptor) is a member of nuclear receptor superfamily transcription factors, belongs to the first subfamily of thyroxine receptors, and is the same as peroxisome proliferator-activated receptor, farnesoid X Receptors, retinol X receptors are closely related. The hormone for liver X receptors is oxysterol, a receptor for cholesterol metabolism. At the same time, it also plays a regulatory role in fat formation, glucose metabolism, immune and inflammatory responses and other links.

Two subtypes of liver X receptors have been distinguished, namely liver X receptor α and liver X receptor β, which are named NR1H3 (LXRα) and NR1H2 (LXRβ) according to the nuclear receptor nomenclature rules. ). Two subtypes of liver X receptor were isolated in 1994 and 1995. The two subtypes of nuclear receptors have the same structure, and 77% of the amino acid sequences in the DNA-binding domain and the ligand-binding domain are identical. Liver X receptor α is mainly expressed in liver tissue cells, but also found in pancreas, spleen and small intestine, while liver X receptor β can be detected in cells throughout the body. In cells, the ligands of liver X receptors are oxides of cholesterol derivatives, and some synthetic compounds such as GW3965 and T0901317 can also be used as ligands to activate liver X receptors. The Chinese name of GW3965 is 3-[3-[[[2-chloro-3-(trifluoromethyl)phenyl]methyl](2,2-biphenylethyl)amino]propoxy]phenylethyl Hydrochloride, screened from triamine compound library; Chinese name of T0901317 is N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1 -Hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide. Currently there is no application of LXR as a target in screening anti-HCV drugs.

Contents of the invention

The purpose of the present invention is to make up for the deficiencies of the prior art, and is to provide an application of a cell target liver X receptor in the preparation of a drug for treating or preventing hepatitis C virus. LXR agonists GW3965 and T0901317 can effectively inhibit the entry of hepatitis C virus within the range of non-toxicity, and can be further developed as drugs for treating or preventing hepatitis C virus infection, and have broad application prospects. The more important application is that it can screen various anti-hepatitis C virus drugs targeting LXR, which provides a new means and approach for the cure and treatment of hepatitis C.

In order to achieve the above-mentioned purpose, the technical scheme adopted in the present invention is:

The application of a liver X receptor as a drug target for treating or preventing hepatitis C virus. The step is to screen a series of compounds for anti-HCV activity, and find that two agonists GW3965 and T0901317 targeting the liver X receptor (LXR) have significant anti-HCV activity, therefore It is confirmed that LXR can be used as an effective target for screening anti-HCV drugs.

The Chinese name of the LXR agonist GW3965 is 3-[3-[[[2-chloro-3-(trifluoromethyl)phenyl]methyl](2,2-biphenylethyl)amino]propoxy ] phenylacetic acid hydrochloride, has the structural formula shown in structural formula I:

Structural Formula I

The Chinese name of LXR agonist T0901317 is N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxyl-1-(trifluoromethyl) Ethyl] phenyl]-benzenesulfonamide has the structural formula shown in structural formula II:

Structural formula II

An application of a cell target liver X receptor in the preparation of a drug for treating hepatitis C virus, the steps of which are as follows:

A. Cytotoxicity experiments of LXR agonists GW3965 and T0901317 on Huh7.5.1: Huh7.5.1 cells were seeded in 96-well cell culture plates at 8×10 ³ cells/well, and after the cells adhered to the wall, 0nM, 100nM, 500nM, 1000nM GW3965 and T0901317 were treated, each group was repeated three wells, placed in a 37°C, 5% CO ₂ incubator for 24 hours, and then treated with 3-(4,5-dimethylthiazole-2)- 2,5-diphenyltetrazolium bromide (thiazolium blue, MTT) method was used to detect the cell viability. The results showed that the CC ₅₀ (median lethal concentration) of GW3965 against Huh7.5.1 was 39.3 μM, and the CC ₅₀ (median lethal concentration) of T0901317 against Huh7.5.1 was 18.1 μM.

B. Evaluation of the anti-HCV activity of LXR agonists GW3965 and T0901317: (1) Construction of plasmid PJFH1-Luc-5AGFP: Plasmid transformation was carried out on PJFH1, and a restriction site XhoI(nt7523) at the C-terminus of the NS5A coding region was selected ~nt7528, aa419~aa420) were inserted into the EGFP gene (amplified from the PEGFP-N1 plasmid). In order to make the hRLuc gene (amplified from the pGL4.70[hRLuc] plasmid) inserted between the 5' NTR and the core gene of the JFH1 genome, a 51nt HCV core gene coding sequence ΔC was added after the 5' NTR to make the recombinant virus The IRES at the 5' end is fully functional; followed by a foot-and-mouth disease virus 2A protein self-cleaving short peptide (foot-and-mouth disease virus 2A, FMDV 2A) fused to the C-terminus of the hRLuc gene. (2) Preparation of viruses JFH1 and JFH1-Luc-5AGFP: Using XbaI linearized plasmids pJFH1 and PJFH1-Luc-5AGFP as templates, viral genome RNA was transcribed in vitro, and then electroporated into Huh-7.5.1 cells. After 9 to 10 days, obvious cytopathic changes will appear, so the culture supernatant is collected and frozen at -80°C after aliquoting. In order to obtain a large amount of virus stock solution, Huh7.5.1 cells were infected with the virus at a multiplicity of infection of 0.02. After obvious cytopathic changes occurred, the infectious supernatant was collected and stored for later use. (3) Huh7.5.1 cells were inoculated in 24-well cell culture plates at 4×10 ⁴ cells/well, cultured in a cell culture incubator at 37°C for 14-18 hours, and the cells were grown into a single layer before use. Dilute GW3965 or T0901317 to 4 concentrations of OnM, 100nM, 500nM, and 1000nM with DMEM medium, with 2 replicates in each group. After different doses of drugs were added to the culture dish for 12 hours, the JFH1-Luc-5AGFP virus was added at a multiplicity of infection of 0.2, and the GFP fluorescence signal was detected 72 hours after infection. Then, the antiviral activity of the drug was studied by observing the change of NS5A-GFP fluorescence intensity and detecting the expression of the virus NS5A gene in situ; (4) Huh7.5.1 cells were seeded in 24-well cell culture plates at ⁴ ×104 cells/well, and kept at 37°C After culturing in the cell incubator for 14 to 18 hours, wait for the cells to grow into a monolayer for later use. Dilute GW3965 or T0901317 into 4 concentrations of OnM, 100nM, 500nM, and 1000nM with DMEM medium, and each group has 3 replicates. After adding different doses of drugs into the culture dish for 12 hours, JFH1-Luc-5AGFP virus was added at a multiplicity of infection of 0.2, luciferase was detected after 72 hours after infection, and then the antiviral activity of the drug was studied by detecting the luciferase activity; ( 5) Seed Huh7.5.1 cells at 2×10 ⁵ cells/well in a 6-well cell culture plate, culture in a cell culture incubator at 37°C for 14-18 hours, and wait for the cells to grow into a single layer before use. Dilute GW3965 or T0901317 into 4 concentrations of OnM, 100nM, 500nM and 1000nM with DMEM medium. After different doses of drugs were added to the culture dish for 12 hours, the wild-type JFH1 virus was added at a multiplicity of infection of 0.2, cell samples were collected, and the expression of HCV non-structural protein NS3 was detected by immunoblotting to study the antiviral activity of the drug. The results showed that the EC ₅₀ (half effective concentration) of GW3965 was 224nM, and the therapeutic index (TI, TI=CC ₅₀ /EC ₅₀ ) was about 175. The EC ₅₀ (half effective concentration) of T0901317 is 78.64nM, and the therapeutic index is about 230. The experimental results show that LXR can be used as an anti-HCV target.

C. Study on the effect of LXR agonists GW3965 and T0901317 on the entry of hepatitis C pseudovirus (HCVpp): 4×10 ⁴ Huh7.5.1 cells were inoculated into 24-well plates, and cultured in a cell incubator at 37°C for 14-18 hours. Wait for the cells to grow into a monolayer for later use. GW3965 or T0901317 was diluted into 4 concentrations of OnM, 100nM, 500nM, and 1000nM with DMEM medium, and each group was repeated three times. The treated Huh7.5.1 cells were infected with VSVpp and HCVpp1a and 1b viruses respectively, and after 72 hours, the luciferase activity was detected with the Luciferase Assay System kit (promega, E1501), and the specificity of the two drugs was studied by detecting the luciferase activity Impact on HCV entry. The results showed that both GW3965 and T0901317 could effectively inhibit the entry of HCVpp in a dose-dependent manner.

D. Study on the combined use of LXR agonists GW3965 and T0901317 with other drugs with anti-HCV activity: Huh7.5.1 cells were seeded in 96-well cell culture plates at 8×10 ³ cells/well, and cultured in a cell culture incubator at 37°C After 14 to 18 hours, wait for the cells to grow into a monolayer and set aside for later use. Cyclosporine A (CsA) or MK-70092-fold gradient dilution was added to the well plate as a control group for separate medication, and each group had 3 repetitions; in addition, 500 nM of GW3965 or GW3965 was added to each dilution concentration. T0901317 was used as the experimental group of combined medication, and each group had three repetitions. After adding the drug, the JFH1-Luc-5AGFP virus was added, and after 72 hours, the cells were lysed, and the effect of GW3965 or T0901317 combined with other drugs was studied by detecting the luciferase activity. The results show that GW3965 and T0901317 can be effectively used in combination therapy not dependent on interferon-alpha.

Liver X receptor is a member of the nuclear receptor superfamily transcription factor, belongs to the first subfamily of thyroxine receptors, and is the same as peroxisome proliferator-activated receptor, farnesoid X receptor, retinoid Alcohol X receptors are closely related. The hormone for liver X receptors is oxysterol, a receptor for cholesterol metabolism. At the same time, it also plays a regulatory role in fat formation, glucose metabolism, immune and inflammatory responses and other links. Two subtypes of liver X receptors have been identified, liver X receptor α and liver X receptor β, which are named NR1H3 (LXR α) and NR1H2 ( LXR β). Two subtypes of liver X receptor were isolated in 1994 and 1995. Liver X receptor α was discovered independently by two research groups, who assigned it the names RLD-1 and LXR, respectively. The two subtypes of nuclear receptors have the same structure, and 77% of the amino acid sequences in the DNA-binding domain and the ligand-binding domain are identical. Liver X receptor α is mainly expressed in liver tissue cells, but also found in pancreas, spleen and small intestine, while liver X receptor β can be detected in cells throughout the body. The liver X receptor can be divided into the following parts: the amino-terminus (N-terminus) with a ligand-independent transcriptional activation domain, the DNA-binding domain with two zinc-finger structures, the Hinge domain capable of binding to DNA, carboxy-terminal (C-terminal) with ligand-binding domain. As an orphan receptor, liver X receptor needs to bind to retinol X receptor (RXR) to form a dimer before it has transcriptional activity. This dimer binds to the liver X receptor response element (LXRE) in the target gene and regulates the transcription of this gene. The liver X receptor response element is a 6-nucleotide repeat sequence separated by 4 nucleotides . In cells, the ligands of liver X receptors are oxides of cholesterol derivatives, and some synthetic compounds such as T0901317 and GW3965 can also be used as ligands to activate liver X receptors. The interaction process between the liver X receptor and the transcription activation gene is divided into three steps: when the concentration of the ligand (hormone) is low, the dimer first binds to the auxiliary repressor to inhibit transcription; after the agonist binds to the dimer The co-repressor breaks away to complete the initial transcription; the co-activator combines with the activated dimer to complete the transcription process. Binding of the liver X receptor or the retinol X receptor to the ligand during transcription can stimulate the activity of the dimer.

Compared with the prior art, the present invention has the following advantages and effects:

1. The therapeutic indices of LXR agonists GW3965 and T0901317 against hepatitis C in vitro were 175 and 230, respectively. This shows that the agonist targeting LXR has good activity against hepatitis C virus.

2. It can inhibit the entry of 1a, 1b, and 2a genotype viruses, has broad-spectrum antiviral activity and has a novel target, and there is no anti-HCV drug that targets entry in clinical practice.

3. Combination of LXR-targeted agonists and other anti-HCV drugs has a good effect. Combined treatment can be carried out in combination with existing anti-hepatitis C virus treatment drugs, and the cure rate of hepatitis C can be increased to more than 80%.

Description of drawings

Figure 1 is a chemical structure formula of LXR agonists GW3965 and T0901317.

Figure 2 is a schematic diagram of the cytotoxicity detection of Huh7.5.1 by LXR agonists GW3965 and T0901317.

Fig. 3 is a schematic diagram of treating Huh7.5.1 cells infected with the virus JFH1-Luc-5AGFP with double reporter genes with LXR agonists GW3965 and T0901317.

The antiviral activity of the drug was studied by observing the change of NS5A-GFP fluorescence intensity and detecting the expression of viral NS5A gene in situ.

Fig. 4 is a schematic diagram of treating Huh7.5.1 cells infected with the virus JFH1-Luc-5AGFP with double reporter genes with LXR agonists GW3965 and T0901317.

Drug antiviral activity was studied by detecting luciferase activity.

Fig. 5 is a schematic diagram of treating Huh7.5.1 cells infected with wild-type virus JFH1 with LXR agonists GW3965 and T0901317.

The antiviral activity of the drug was studied by detecting the expression of HCV nonstructural protein NS3 by immunoblotting.

Fig. 6 is a schematic diagram of treating Huh7.5.1 cells infected with HCV pseudovirus system (HCVpp) and VSV pseudovirus system (VSVpp) with LXR agonists GW3965 and T0901317, respectively.

The specific effect of these two drugs on HCV entry was studied by detecting luciferase activity.

Fig. 7 is a schematic diagram for detecting the effect of combining LXR agonists GW3965 and T0901317 with other drugs.

Detailed ways

In order to better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.

The cell culture model is the most commonly used screening model. Its advantages are: it can provide a large number of cells with the same genetic traits as the research object, it is easy to operate, can eliminate the influence of other external factors, and can detect the effective concentration and therapeutic index of the drug. Mechanistic studies provide more basis. For many years after the identification of HCV in 1989, the development of HCV cell culture systems was very slow. There are two main reasons: one is the difficulty in molecular cloning of HCV genome; the other is the lack of cell lines that support HCV infection and replication. After more than 20 years of development, at present, there are mainly three HCV cell culture systems: HCV infectious clone system (infectious HCV cell culture system), HCV pseudovirus system (HCV pseudoparticle system) and HCV replica system (HCV replica system). The present invention adopts the above three systems to respectively verify the effects of LXR agonists GW3965 and T0901317 on HCV infection, entry and RNA replication.

Example 1:

Evaluation of the anti-hepatitis C virus activity of the LXR agonists GW3965 and T0901317.

1. Experimental materials

1.1 Cells, plasmids, viruses and drugs

Huh7.5.1 cells were donated by Dr.F.V.Chisari; plasmid pJFH1 containing the complete genome sequence of HCV type 2a JFH1 strain was donated by Prof. Dr. Takaji Wakita; plasmid pEGFP-N1 was purchased from Clontech; plasmid pGL4.70[hRLuc] was purchased from promega company; the virus JFH1-Luc-5AGFP with double reporter genes was prepared by our laboratory; GW3965 and T0901317 were purchased from sigma company.

1.2 Reagents

DMEM medium was purchased from GIBCO Company; Renilla luciferase detection kit was purchased from Promega Company; anti-HCV NS3 mouse monoclonal antibody was purchased from Henan Bioengineering Technology Research Center; anti-GAPDH mouse monoclonal antibody was purchased from Beijing Zhongshan Golden Bridge Company; HRP-labeled goat anti-mouse secondary antibody was purchased from Thermo Company; restriction endonuclease was purchased from NEB Company.

1.3 Experimental Instruments

20/20检测仪为promega公司产品；Mini-PROTEAN垂直蛋白电泳设备购自BioRad公司；VE-186转移电泳槽购自上海天能科技有限公司。Zeiss advanced inverted microscope Axio observer A1 (Carl Zeiss); the chemiluminescence detector is the FluorChem HD2 system from Alpha Innotech;

The 20/20 detector is a product of Promega; the Mini-PROTEAN vertical protein electrophoresis equipment was purchased from BioRad; the VE-186 transfer electrophoresis tank was purchased from Shanghai Tianneng Technology Co., Ltd.

2. Experimental methods and results

2.1 Drug Cytotoxicity Detection

Cultured in a humidified incubator at 37°C with 5% CO2. A DMEM medium containing 10% FBS, 100 U/mL of penicillin and streptomycin was used. The cells were subcultured after reaching 90% confluence, and the subculture ratio was 1/4-1/6. Huh7.5.1 cells were seeded in a 96-well cell culture plate at 8×10 ³ cells/well, and the cells were attached to the wall for later use; the drug was serially diluted with the medium with an initial concentration of 10 mM, and each gradient had 3 replicate wells. After culturing for 72 hours, add 20 μl of 5 mg/ml MTT to each well, and place it in a cell culture incubator to continue culturing; after culturing for 4 hours, discard the supernatant of the culture medium, and add 100 μl/well triple dissolving solution to each well (the dissolving solution consists of 10 g of SDS, isobutyl Alcohol 5ml, 10M HCl 0.1ml, dissolved in double distilled water to make 100ml), dissolved overnight in a 37°C incubator, then the enzyme-linked detector detects the absorbance at 570nm wavelength, corrects the wavelength to 630nm, and calculates the cell survival rate of each drug concentration .

The results showed (Figure 2): the CC ₅₀ of GW3965 was 39.3 μM, and the CC ₅₀ of T0901317 was 18.1 μM, indicating that they have a relatively safe application range.

2.2 Construction of plasmid pJFH1-Luc-5AGFP and preparation of virus JFH1 and JFH1-Luc-5AGFP:

Plasmid transformation was performed on PJFH1, and a restriction site XhoI (nt7523-nt7528, aa419-aa420) at the C-terminus of the NS5A coding region was selected and inserted into the EGFP gene. In order to insert the hRLuc gene between the 5'NTR and the core gene of the JFH1 genome, a 51nt HCV core gene coding sequence ΔC was added after the 5'NTR to make the 5' end IRES function of the recombinant virus complete; followed by a foot-and-mouth disease Virus 2A protein self-cleavage short peptide (foot-and-mouth disease virus 2A, FMDV 2A) is fused to the C-terminus of hRLuc gene. After the plasmid was successfully constructed, it was sequenced and identified. Using XbaI-linearized plasmids pJFH1 and PJFH1-Luc-5AGFP as templates, viral genomic RNA was transcribed in vitro, and then electroporated into Huh-7.5.1 cells. After 9 to 10 days, obvious cytopathic changes will appear, so the culture supernatant is collected and frozen at -80°C after aliquoting. In order to obtain a large amount of virus stock solution, Huh7.5.1 cells were infected with the virus at a multiplicity of infection of 0.02. After obvious cytopathic changes occurred, the infectious supernatant was collected and stored for later use. The titer of the obtained virus supernatant was about 10 ⁵ ffu/ml.

2.3 Observe the positive rate of GFP to detect in situ the activity of LXR agonist GW3965 and T0901317 anti-virus strain JFH1-Luc-5AGFP;

The virus JFH1-Luc-5AGFP with dual reporter genes has a GFP fluorescent tag at the C-terminus of the HCV nonstructural protein NS5A, and the infection and replication of HCV in cells can be observed in real time with a fluorescence microscope in living cells. Huh7.5.1 cells were inoculated in 24-well cell culture plates at 4×10 ⁴ cells/well, cultured in a cell culture incubator at 37°C for 14-18 hours, and the cells were grown into a single layer before use. Dilute GW3965 or T0901317 to 4 concentrations of OnM, 100nM, 500nM, and 1000nM with DMEM medium, with 2 replicates in each group. GW3965 or T0901317 was diluted with DMEM medium into different doses and added to the culture dish. After 12 hours, JFH1-Luc-5AGFP was added at a multiplicity of infection of 0.2. GFP fluorescence signal was detected 72 hours after infection. In order to calibrate the GFP fluorescence signal with the number of cells and observe the sublocalization of NS5A-GFP in the cells, the applicants used DAPI to perform nuclear staining experiments. First discard the supernatant of the culture medium, fix the cells with fixative solution at room temperature (20-25°C, the same below) for 30min; then wash with PBS (pH7.4) 3 times, 5min each time; then add DAPI diluted in binding solution for staining The solution was incubated at room temperature for 30 minutes; finally washed with PBS (pH7.4) for 3 times, each time for 5 minutes; then the fluorescent signal was observed under an ordinary inverted fluorescence microscope. The photos of GFP-positive cells taken under a fluorescence microscope are a representative group selected from photos of multiple different fields of view.

The results showed (Fig. 3): In the control group treated with DMSO without adding drugs, there was obvious GFP fluorescence signal, and the number of cells was quantified by DAPI staining, and the average number of positive cells expressing GPF in each field of view was about 40-40. 50%; compared with the control, after drug treatment, the ratio of GFP-positive cells decreased significantly, and showed a certain dose-dependent effect. When treated with 100 nM of GW3965 or T0901317, the ratio of GFP positive cells was reduced to about 15%. When the drug concentration increased to 1 μM, the rate of GFP-positive cells after the treatment of the two drugs dropped to less than 5%. The experimental results show that LXR agonists GW3965 and T0901317 have good antiviral activity.

2.4 The activity of LXR agonists GW396 and T09013175 against virus strain JFH1-Luc-5AGFP was verified by detecting luciferase activity.

The luciferase reporter gene system uses luciferin as a substrate to detect luciferase activity. Luciferase can catalyze the oxidation of luciferin to oxidized luciferin, and in the process of luciferin oxidation, bioluminescence will be emitted. The bioluminescence released during the oxidation of fluorescein can then be measured by a fluorometer, also known as a luminometer or a liquid scintillation meter. Luciferin and luciferase, a bioluminescent system, can detect gene expression extremely sensitively and efficiently. The JFH1-Luc-5AGFP virus strain prepared by the applicant's laboratory has a Luciferase reporter gene, so the influence of drugs on viral gene expression can be quantified sensitively. Huh7.5.1 cells were inoculated in 24-well cell culture plates at 4×10 ⁴ cells/well, cultured in a cell culture incubator at 37°C for 14-18 hours, and then used after the cells grew into a monolayer. Dilute GW3965 or T0901317 with culture medium to 4 concentrations of OnM, 100nM, 500nM, and 1000nM, with 3 replicates in each group. After different doses of drugs were added to the culture dish for 12 hours, JFH1-Luc-5AGFP virus was added at a multiplicity of infection of 0.2, luciferase was detected 72 hours after infection, the cells to be tested were washed twice with PBS, and then detected with Renilla luciferase According to the kit instructions, add lysate to fully lyse the cells, then add the lysate to the diluted substrate, and perform detection on a luciferase detector. The results are displayed in relative light units (RLU). Experimental data are expressed as the mean of three independent experiments, and errors are expressed as standard deviations.

The results showed ( FIG. 4 ): setting the average fluorescence value of the positive control group treated with DMSO without drugs as 100%, the relative value of luciferase activity after GW3965 or T0901317 treatment decreased significantly with the increase of drug dose. Analysis by SPSS software: the EC50 of GW3965 is 224nM, and the EC50 of T0901317 is 78.64nM. According to their corresponding CC ₅₀ values, their therapeutic index (TI) is calculated to be 175 and 230, respectively.

2.5 The activity of LXR agonists GW3965 and T0901317 against virus strain JFH1 was verified by Western blotting.

Huh7.5.1 cells were seeded in 6-well cell culture plates at 2×10 ⁵ cells/well, cultured in a cell culture incubator at 37°C for 14-18 hours, and the cells were grown into a single layer before use. Dilute GW3965 or T0901317 into 4 concentrations of OnM, 100nM, 500nM and 1000nM with DMEM medium. After adding different doses of drugs into the culture dish for 12 hours, the JFH1 virus was added at a multiplicity of infection of 0.2, and the cells were harvested 72 hours after infection for immunoblotting detection. After rinsing the cells once with PBS, the cells were pipetted and collected into EP tubes with 1ml/well of PBS. After lysing with the lysate containing protease inhibitors, the sample buffer was added in proportion to perform polyacrylamide Gel electrophoresis separates and arranges protein molecules according to molecular weight. Then the protein molecules were transferred to polyvinylidene fluoride (PVDF) membrane by electroporation. After blocking with 5% (mass:volume) skimmed milk, the primary antibody was added to bind overnight at 4°C. Rinse with TBST for 3 times, each time for 5 min, to elute non-specific binding, then add the corresponding secondary antibody to bind, rinse with TBST for 3 times, and perform substrate color detection.

The results showed ( FIG. 5 ): compared with the control group treated only with DMSO, the expression of HCV non-structural protein NS3 was significantly reduced after GW3965 or T0901317 treatment, and the effect was dose-dependent. In addition, the immunoblotting brightness of the cell housekeeping gene GAPDH was basically the same, indicating that a relatively consistent number of cells was maintained during loading. Therefore, the results showed that LXR agonists GW3965 and T0901317 significantly inhibited the expression of HCV NS3 gene and had significant anti-HCV JFH1 viral activity. This shows that LXR can be used as a target to screen anti-HCV drugs.

Example 2: Study on the Effect of LXR Agonist GW3965 and T0901317GW3965 on the Entry of Hepatitis C Pseudovirus (HCVpp)

1. Experimental materials

1.1 Cells, viruses and drugs

Huh7.5.1 cells; GW3965 and T0901317; plasmid PNL4.3-R-E-Luc and plasmid pVpack-VSV-G containing the VSV viral envelope protein were obtained from the AIDS Research Division of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The plasmids containing HCV1a and 1b envelope proteins were donated by Mr. Qi Zhongtian.

1.2 Reagents

DMEM medium was purchased from GIBCO; transfection reagent Lipofectamine2000 was purchased from Invitrogen; Firefly luciferase detection kit was purchased from Promega.

1.3 Experimental Instruments

20/20检测仪为promega公司产品。

The 20/20 detector is a product of Promega.

2. Experimental methods and results

2.1 Compared with the live virus, the HCV pseudovirus has similar cell infection characteristics, which can be used to simulate the early entry process of the true virus infected cells, and the reporter gene (EGFP, luciferase, etc.) is carried in the pseudovirus, which can quickly and conveniently carry out various detection. HCV pseudoviruses are viral particles obtained by packaging the genome of another virus with the HCV envelope glycoprotein. The pseudovirus used in this experiment was co-transfected into 293T cells by expressing the full-length HCVE1E2 gene and the non-enveloped HIV-1 proviral genome (pNL4.3.Luc.RE-) containing the reporter gene, and collecting the supernatant Purified by ultracentrifugation. Inoculate 4×10 ⁴ Huh7.5.1 cells into a 24-well plate and wait for the cells to adhere to the wall for use. GW3965 or T0901317 was diluted into 4 concentrations of OnM, 100nM, 500nM, and 1000nM with DMEM medium, and each group was repeated three times. The treated Huh7.5.1 cells were infected with VSVpp and HCVpp1a, 1b viruses, and the luciferase activity was detected with Luciferase Assay System kit 72 hours later.

The results showed ( FIG. 6 ): GW3965 and T0901317 could effectively inhibit the entry of HCVpp in a dose-dependent manner, which indicated that the anti-HCV activity of GW39655 and T0901317 was based on the inhibition of HCV viral entry. GW3965 and T09013175 had no significant effect on the entry of VSV pseudoviruses, indicating that their inhibitory effect on HCV entry was specific. Moreover, these two drugs have inhibitory effects on the entry of HCV1a and 1b pseudoviruses, indicating that GW39655 and T0901317, as HCV virus entry inhibitors, have no genotype selectivity. And it shows that drugs targeting LXR act on the entry stage in the life cycle history of HCV.

Example 3: Combination study of LXR agonists GW3965 and T0901317 with other drugs with anti-HCV activity.

1. Experimental materials

1.1 Cells, viruses and drugs

Huh7.5.1; virus JFH1-Luc-5AGFP; GW3965 and T0901317; CsA (purchased from Sigma Company) and MK-7009 (purchased from Zannan Company).

1.2 Reagents

DMEM medium was purchased from GIBCO; Renilla luciferase detection kit was purchased from Promega.

1.3 Experimental Instruments

The 20/20 detector is a product of Promega.

2. Experimental methods and results

2.1 Inoculate Huh7.5.1 cells in 96-well cell culture plates at 8×10 ³ cells/well, culture in a cell culture incubator at 37°C for 14-18 hours, and wait for the cells to grow into a single layer before use. Cyclosporine A (CsA) or MK-70092-fold gradient dilution was added to the well plate as a control group for separate medication, and each group had 3 repetitions; in addition, 500 nM of GW3965 or GW3965 was added to each dilution concentration. T0901317 was used as the experimental group of combined medication, and each group had three repetitions. After adding the drug, the JFH1-Luc-5AGFP virus was added, and after 72 hours, the cells were lysed, and the effect of GW3965 or T0901317 combined with other drugs was studied by detecting the luciferase activity.

The results showed (Figure 7): cyclosporine A (CsA) inhibited the replication of HCV by antagonizing the promotion of RNA replication by the HCV host factor cyclophilin A; All have better anti-HCV virus activity. When combined with GW3965 or T0901317, there is very obvious antiviral activity at very low concentrations of CsA or MK-7009. The results show that GW3965 or T0901317 can be effectively used in combination therapy not dependent on alpha interferon. This result also shows that drugs targeting LXR can be used in combination with other anti-HCV drugs.

Claims (1)

1. the application of cell target spot liver X receptor in preparation treatment or prevention of hepatitis C medicine.

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