CN116785287A - Application of deubiquitinase inhibitor in preparation of drugs for inhibiting replication of hepatitis B virus by enhancing interferon - Google Patents

Abstract

The invention discloses the use of deubiquitinating enzyme inhibitors in preparing drugs that inhibit hepatitis B virus replication by enhancing interferon. The present invention uses deubiquitinating enzyme inhibitors to enhance interferon to inhibit viral replication to treat chronic hepatitis B, and verifies that deubiquitinating enzyme inhibitors enhance the antiviral effect of interferon signaling pathways to enhance Interferons inhibit viral replication.

Description

Preparation of deubiquitinating enzyme inhibitors inhibits hepatitis B virus replication by enhancing interferon Applications in medicines

Technical field

The present invention relates to the application of deubiquitinating enzyme inhibitors in the preparation of drugs that inhibit hepatitis B virus replication by enhancing interferon, and belongs to the field of medical technology.

Background technique

The latest global hepatitis report from the World Health Organization (WHO) shows that 257 million people around the world are still infected with HBV. HBV infection is divided into acute and chronic infections. Chronic infection can cause chronic hepatitis B (CHB), cirrhosis ( LC) and primary hepatocellular carcinoma (HCC) and other related diseases, HCC caused by chronic HBV infection ranks fifth in the world. There are currently two main categories of first-line drugs clinically used to treat CHB, including nucleoside analogues (NA) and interferon α (IFNα). Among them, NAs (including lamivudine, adefovir dipivoxil, and entecavir and tenofovir) can effectively inhibit viral replication, but it is difficult to completely eliminate HBV. There are problems such as viral drug-resistant mutations and rebound after drug withdrawal. Patients often need to take long-term or even lifelong medication, and nucleoside analog therapy can only be used in a very small number of patients. To achieve "clinical cure" of the disease. CHB patients treated with polyethyl alcohol interferon α (PEG-IFNα) can achieve higher HBeAg and HBsAg seroconversion rates, and can induce sustained antiviral responses in about 30% of patients who discontinue treatment.

Interferon (IFN) is a type of secreted protein with multiple biological activities and plays an important regulatory role in the body's innate immunity, anti-virus and anti-tumor. IFN can exert antiviral effects in multiple stages of the viral replication cycle and is widely used in clinical antiviral treatment, such as the treatment of CHB caused by HBV infection. Antiviral innate immunity mediated by the IFN signaling pathway is an important part of the host's resistance to pathogen invasion. After activation of this pathway, it induces the expression of hundreds of ISGs. These gene products target different aspects of viral replication and initiate the antiviral innate immune response. Resist virus infection and jointly establish the host's "anti-viral" state.

Antiviral natural immunity is the host's first line of defense against viral infection. When a virus invades a host cell, pathogen-related molecular patterns such as viral nucleic acids will be recognized by intracellular pattern recognition receptors, thereby activating a series of intracellular signaling cascades. response, inducing the expression of downstream interferon-α (IFN-α) and other type I IFNs as well as pro-inflammatory factors, thereby initiating the anti-viral natural immune response to resist viral infection. Type I IFN is produced and binds to the IFN receptor on the cell surface, thereby activating the downstream Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway, inducing the expression of a large number of interferon-stimulated genes (ISGs), and during viral replication. Multiple stages of the cycle exert antiviral effects, but the clinical response efficiency of IFNα in treating CHB is not high.

Ubiquitination is a post-translational modification that covalently connects the carboxyl group at the C-terminus of the ubiquitin molecule to the N-terminus of the substrate protein or the amino group of lysine through a cascade reaction catalyzed by ubiquitin ligase. Ubiquitination can change its function, localization, stability and protein-protein interactions. Deubiquitination is the opposite process to ubiquitination. Under the catalysis of deubiquitinases (DUBs), the ubiquitin molecules on the substrate protein are removed, thereby avoiding degradation by the proteasome. . Inhibiting deubiquitinating enzymes can lead to selective degradation of proteins. DUBs-mediated deubiquitination modification plays a very important regulatory role in antiviral innate immunity, especially in the production or effector pathways of interferons, and can be positive or negative. To regulate the antiviral effect of interferon.

Contents of the invention

The invention provides the application of a deubiquitinating enzyme inhibitor in the preparation of a drug that inhibits hepatitis B virus replication by enhancing interferon. Deubiquitinating enzyme inhibitors can play a therapeutic role by enhancing interferon to inhibit hepatitis B virus replication.

In order to achieve the above object, the technical solution adopted by the present invention is the application of deubiquitinating enzyme inhibitors in the preparation of drugs that inhibit hepatitis B virus replication by enhancing interferon.

In some embodiments of the present invention, the deubiquitinating enzyme inhibitor is GEN-6776 and AZ1, or a pharmaceutically acceptable salt of both.

In some embodiments of the present invention, the interferon enhanced by the deubiquitinating enzyme inhibitor is type I IFN. The deubiquitinating enzyme inhibitor enhances the antiviral effect of the interferon signaling pathway, thereby enhancing the interferon to inhibit the replication of hepatitis B virus.

Furthermore, the GEN-6776 and AZ1 deubiquitinating enzyme inhibitors enhance the antiviral effect of interferon alpha signaling pathway, thereby enhancing interferon alpha to inhibit the replication of hepatitis B virus. The hepatitis B is chronic hepatitis B.

The present invention uses deubiquitinating enzyme inhibitors to enhance interferon's inhibition of hepatitis B virus replication to play a therapeutic role, and verifies that deubiquitinating enzyme inhibitors enhance the antiviral effect of interferon signaling pathways to enhance interferon inhibition. Replication of viruses. The present invention verifies that the deubiquitinating enzyme inhibitors GEN-6776 and AZ1 are particularly effective in enhancing interferon α to inhibit the replication of hepatitis B virus, and can be used to prepare drugs for the treatment of chronic hepatitis B.

Description of the drawings

Figure 1 shows the activation effects of different deubiquitinating enzyme inhibitors on interferon-induced ISRE activity in Example 2 of the present invention;

Figure 2 shows the activation effects of different deubiquitinating enzyme inhibitors on interferon-induced ISG in Example 2 of the present invention;

Figure 3 shows the effect of GEN6776 and AZ1 on enhancing the effect of interferon α on inhibiting HBV replication in Example 3 of the present invention.

Detailed ways

In order to better understand the essence of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

The principle of the present invention is that deubiquitinating enzyme inhibitors can treat chronic hepatitis B by enhancing interferon and inhibiting hepatitis B virus replication. Specifically, deubiquitinating enzyme inhibitors GEN-6776 and AZ1 are positively regulated It inhibits the JAK-STAT signaling pathway of type I interferon, enhances the antiviral effect of type I interferon, and can significantly promote the antiviral effect of interferon during HBV replication and transfection. Therefore, GEN-6776 and AZ1 can be used to prepare and treat chronic Drugs for hepatitis B infection can also be used to inhibit the replication of other viruses that strongly induce type I interferon production, such as vesicular stomatitis virus (VSV). The type I interferons include IFNα and IFNβ.

The deubiquitinase inhibitors GEN-6776 and AZ1 used in the examples of the present invention are both from Selleck Company, CAS: 2009273-71-4 and 2165322-94-9.

Example 1

1. Toxicity of deubiquitinating enzyme inhibitors to hepatocytes.

1. Materials and methods

1.1. Instruments: _CO2 incubator, small high-speed centrifuge, ultracentrifuge, microplate reader, Real-Time System instrument.

1.2. Reagents and consumables: liver cancer cell line Huh7 cells (derived from ATCC), fetal calf serum (BI Company), DMEM culture medium, sodium pyruvate and penicillin-streptomycin (Hyclone Company), IFNα-2a (MCE Company, United States) , trypsin (GIBCO, USA), 3000Reagent transfection reagent (Thermo, USA), cell proliferation detection kit (CellTiter/> AQueous Non-radioactive Cell Proliferation Assay) (Promega Company), SYBR (Roche Company, USA), Proteinase K (Roche Company, USA), microtubule enzyme (NEB Company, USA) and deubiquitinating enzyme inhibitors. The deubiquitinating enzyme inhibitors used are shown in Table 1.

1.3.Method:

1.3.1. Cell culture

Liver cancer cell Huh7 was cultured in DMEM cell culture medium containing 10% FBS and 1% PS.

1.3.2. MTT method is used to detect the effects of various drugs and compounds on cytotoxicity.

Inoculate 4.0×10 ³ Huh7 cells in a 96-well plate, add different concentrations of compounds, add MTS/PML mixture after 72 hours of culture, and place in a 37°C incubator for 2 hours before measuring the absorbance at 490 nm.

2. Results

The present invention uses the MTT method to detect the cytotoxicity of 10 μM deubiquitinating enzyme inhibitor to hepatocyte Huh7. The results are shown in Table 1. Among them, SJB2-043, b-AP15, PR-619, NSC632839, EOAI3402143 and GSK2643943A have high cytotoxicity, while GEN-6776, TCID, ML364 and AZ1 are relatively less toxic in liver cells Huh7.

Table 1 Deubiquitinating enzyme inhibitors

(Cytotoxicity is defined as a significant decrease in cell viability of the compound at a concentration of 10 μM measured by MTT assay: a decrease of 75% is +++)

Example 2

1. Preparation and detection of HBV DNA

1. Extraction of total RNA from Huh7 cells

1.1. Discard the culture medium from the six-well plate, add 500uL PBS buffer solution, rinse gently, discard, add 500uL reagent TRIzol, and shake for 5 minutes at room temperature for lysis;

1.2. Beat until no longer sticky, transfer to EP tube, try to avoid bubbles (can be stored at -80℃);

1.3 Add 100uL of chloroform, shake for 15 seconds, and put on ice for 10 minutes;

1.4. Centrifuge at 4°C for 15 minutes at a speed of 12000g, and suck 190uL of the supernatant transparent layer into another 1.5mL EP tube;

1.5. Add 190uL isopropyl alcohol for precipitation, vortex for 15 seconds, and place on ice for 10 minutes;

1.6. Centrifuge at 12000g for 10 minutes at 4°C, discard the supernatant, and blot as dry as possible;

1.7. Add 1ml of 75% ethanol, centrifuge at 4°C for 5 minutes at a speed of 7500g;

1.8. Air dry for 5-10 minutes, add 15uL DEPC water to dissolve, shake and mix, then centrifuge, and measure the RNA concentration;

1.9. Use nucleic acid electrophoresis to detect whether the RNA is degraded. If not, perform reverse transcription or store it at -80°C.

2. Reverse transcription of RNA

2.1. Remove the RNA from the sample (total system 10uL): The reagents are prepared according to the following system, mix well and then evaporate:

Reverse transcription reagent 1 (IuL), reverse transcription reagent 2 (2uL), RNA sample (1ng) corresponding volume, enzyme-free water to make up the total system to 10ul, reaction conditions: 42℃, 2min≧10℃, hold.

2.2. Reverse transcription (total system 20uL), mix the reagents according to the following system and then isolate: 10uL mixture, reverse transcription reagent 3 (1ul), reverse transcription reagent 4 (4ul), reverse transcription reagent 5 (1ul), reverse transcription Reagent 6 (4ul).

3. Extraction of viral DNA and detection of HBV DNA amount

HBV DNA in viral core particles was extracted using sucrose density gradient centrifugation: 3 days after transfection, the cells were lysed with CPLB lysis buffer (10mmol/LTris-HCl, 1mmol/L EDTA, 1% NP-40, 2% sucrose), 10000r /min for 5 min, the supernatant was digested with microtubule enzyme (Micrococcal Nuclease, M'Nase) at 37°C for 1 h, and then ultracentrifuged at 45 000 r/min for 2 h in 30% sucrose density medium to extract virus particles, and then digested with proteinase K overnight. Use phenol/chloroform extraction and ethanol precipitation to obtain HBV DNA. Use fluorescence quantitative PCR to detect HBV DNA: 10ul system, 2×SYBR 5ul, HBV primers FP384 (5'-TGCGGCTTTTTATCATTCC-3'), RP438 (5'-ATACCTTGGTAGTCCAGAAGAACCA-3') 0.25ul each, RNAse free water 3ul, HBV DNA 1ul . A real-time fluorescence quantitative nucleic acid amplification detection system was used to detect the amount of HBV DNA.

2. The activation effect of deubiquitinating enzyme inhibitors on interferon-induced ISRE and ISG activities.

1. Effect of deubiquitinating enzyme inhibitors on interferon-induced ISRE activity

According to the results of the cytotoxicity experiment in Example 1, deubiquitinating enzyme inhibitors (GEN-6776, TCID, ML364 and AZ1) that are non-toxic to cells at 10 μM were selected for interferon-induced ISRE activity detection: 5 μM of the above Huh7.0 cells were treated with deubiquitinase inhibitors and DMSO and stimulated with exogenous IFNα, and then the activity of ISRE was detected by dual-luciferase assay.

2. Dual luciferase assay

Prepare cell lysis solution 1x PLB (100uL/well) in advance, place it in a 24-well plate at room temperature, discard the culture medium, rinse gently with PBS buffer solution, discard the PBS, add 1x PLB, place on a shaker, lyse cells at room temperature for 30 minutes, each well 50uL of LARII is required. Calculate the required amount and thaw it in advance. Each well of Stop&Glo is also 50uL and needs to be prepared in advance. Both Stop&Glo and LARII are strictly protected from light. Perform fluorescence measurement in a light-proof environment: Add LARI to 20uL of cell lysis solution and pipette. After mixing, detect, which is the firefly luciferase value; then add 50uL Stop&Glo, mix in the same manner, and read, which is the Renilla luciferase value; process the data and perform statistical analysis.

3. Effects of deubiquitinating enzyme inhibitors on the expression levels of ISGs

After Huh7.0 cells were treated with the above-mentioned deubiquitinating enzyme inhibitors at a concentration of 5 μM and induced with IFNα, the transcription levels of A3G and SAMHD1 were detected by qRT-PCR.

4. Results

As can be seen from Figure 1, deubiquitinase inhibitors GEN-6776, ML364 and AZ1 can significantly enhance IFNα-induced ISRE activity, indicating that they may be positive regulatory compounds that promote the antiviral effect of IFNα, while TCID has an effect on interferon α-induced There was no significant enhancement of ISRE activity.

As shown in Figure 2, under IFNα induction, the deubiquitinase inhibitors GEN-6776 and AZ1 can significantly enhance the expression of ISGs such as A3G and SAMHD1, indicating that the two deubiquitinase inhibitors GEN-6776 and AZ1 are The expression of ISGs, the downstream effector molecules that regulate the IFN signaling pathway, may promote the antiviral effect of IFNα.

In summary, since two deubiquitinating enzyme inhibitors, GEN-6776 and AZ1, can significantly enhance the IFNα-induced ISRE promoter activity and increase the expression of downstream antiviral effector molecules ISGs such as SAMHD1 and A3G, GEN-6776 and AZ1 can positively promote the type I interferon JAK-STAT signaling pathway, are positive regulatory drugs that promote the antiviral effect of IFNα, and can be used as targets for the treatment of HBV infection.

Example 3

Huh7.0 cells were treated with GEN-6776, ML364 and AZ1 at a working concentration of 5 μM. After transfection with HBV replication plasmid, they were treated with IFNα (1000IU/ml) for 3 consecutive days. HBeAg, HBsAg and intracellular viruses in the cell supernatant were detected. HBV-DNA levels in particles.

As shown in Figure 3, after IFNα treatment, GEN-6776 and AZ1 significantly reduced the levels of viral HBeAg and HBsAg in the cell supernatant and the level of HBV DNA in intracellular viral core particles. In other words, GEN-6776 and AZ1 promote the effect of IFNα on inhibiting HBV replication.

The above results indicate that GEN-6776 and AZ1 promote the inhibitory effect of interferon on HBV by positively regulating the IFN-induced JAK-STAT signaling pathway, thereby reducing intracellular HBV levels and achieving the treatment of chronic hepatitis B.

The above are only examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the pending application. within the scope of the claims.

Claims (5)

1. Application of deubiquitinating enzyme inhibitors in the preparation of drugs that inhibit hepatitis B virus replication by enhancing interferon. 2. The application according to claim 1, characterized in that: the deubiquitinating enzyme inhibitor is GEN-6776 or AZ1, or a pharmaceutically acceptable salt of both. 3. The application according to claim 2, characterized in that: the interferon enhanced by the deubiquitinating enzyme inhibitor is type I IFN. 4. Application as claimed in claim 3, characterized in that: the GEN-6776 and AZ1 deubiquitinating enzyme inhibitor enhance the antiviral effect of interferon alpha signaling pathway, thereby enhancing the inhibition of hepatitis B virus by interferon alpha. copy. 5. The application according to claim 1, characterized in that: the hepatitis B is chronic hepatitis B.