Application of G quadruplex ligand and entecavir combined drug in resisting HBV (Hepatitis B Virus) infection

Abstract

The invention discloses an application of a G quadruplex ligand and entecavir combined drug in resisting HBV (hepatitis B virus) infection, and relates to the technical field of HBV treatment drugs. The invention determines that a G quadruplex sequence and a G quadruplex structure exist in an HBV genome, G quadruplex ligands TMPyP4 and BRACO19 can be combined with the HBV G quadruplex structure and inhibit transcription and translation of HBV, and TMPyP4 and existing clinical drug ETV combined medication have obvious synergistic effect, which indicates that the HBV G quadruplex can be used as a new drug target for resisting virus infection. The results of the invention can provide new tools and ideas for clinical prevention and treatment of hepatitis B, especially have broad prospects in the aspects of development, clinical application and the like of anti-hepatitis B drugs and vaccines, and can also be directly applied to the field of scientific research or guide the development of inhibitors for inhibiting HBV life cycle. The invention has important application value for obtaining new anti-HBV drug targets and screening new drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of HBV therapeutic drugs, and in particular to the application of a G-quadruplex ligand combined with entecavir in anti-HBV infection. Background Art

[0002] Viral hepatitis Viral hepatitis is currently the most serious infectious disease in China. There are currently about 70 million hepatitis B virus carriers in my country, accounting for about 1 / 3 of the global number. In 2020, the number of hepatitis B cases in China was 902,476, and the total number of HBV infections and cases ranked first in the world. 80% of liver cancer in my country is related to hepatitis B, and a considerable number of chronic hepatitis B patients further develop cirrhosis and liver cancer. Therefore, HBV infection is an important cause of cirrhosis and liver cancer in my country.

[0003] At present, the clinical treatment methods for chronic hepatitis B patients are mainly divided into two categories according to the mechanism. One category is nucleoside analogs, which directly act on the virus itself by inhibiting reverse transcription from pgRNA to rcDNA, and dominate the clinical HBV antiviral treatment because of its convenience and efficiency. Although HBV has vaccines and drugs, it cannot be cured. NAs treatment rarely cures HBV (defined as HBsAg clearance or seroconversion). The other category is interferon, which acts on the host immune system and aims to achieve functional treatment of HBV by enhancing the host immune system. However, for most patients with chronic hepatitis B, clearing and curing HBV is still an unrealized goal. Therefore, it is of great significance to find new therapeutic targets for the conserved sequences of the HBV genome. Summary of the invention

[0004] The present invention provides an application of a G-quadruplex ligand combined with entecavir in anti-HBV infection. By combining a G-quadruplex ligand with entecavir (ETV), it is found that the replication of HBV virus can be significantly inhibited. This is specifically achieved through the following technologies.

[0005] In a first aspect, the present invention provides an application of a G-quadruplex ligand in combination with entecavir in the preparation of a drug for anti-HBV infection. The G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

[0006] Furthermore, the G-quadruplex ligand is TMPyP4.

[0007] Furthermore, the molar ratio of the G-quadruplex ligand TMPyP4 and entecavir is (0.2-1):1;

[0008] Specifically, the molar ratio of the G-quadruplex ligand TMPyP4 and entecavir is 1:1.

[0009] Within the non-cytotoxic concentration range verified by the CCK8 experiment, the AD38 cell line can produce HBV virus. The present invention adds different concentrations of TMPyP4 and BRACO19 to the cell culture medium, co-cultures with the AD38 cell line for 48 hours, and uses a western blot experiment to detect the cell HBc protein level. It is found that as the ligand concentration increases, the level of HBc protein decreases, which means that the ligands TMPyP4 and BRACO19 inhibit the production of HBV virus. In the cell model of HBV infection NTCP, the present invention also verifies that the addition of ligands TMPyP4 and BRACO19 can effectively reduce the content of HBs and HBe proteins in the cell culture medium supernatant, which is statistically significant compared with the control group. This shows that the ligands TMPyP4 and BRACO19 have a therapeutic effect against HBV infection.

[0010] The present invention uses four G-quadruplex ligands, TMPyP4, BRACO19, PDS, and PDP, alone or in combination with entecavir (ETV) to find that when used alone, TMPyP4 and BRACO19 can effectively inhibit HBV replication, among which TMPyP4 has a better therapeutic effect. When TMPyP4 and ETV are used alone, the inhibition rate of HBV virus level is about 50%, and when the two are used in combination, the inhibition rate is significantly increased to about 72%. Compared with the use of a single drug, the therapeutic effect of the combined drug is significantly improved, and the replication of HBV virus can be significantly inhibited.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for preventing HBV infection, which is composed of a G-quadruplex ligand and entecavir. The G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

[0012] Furthermore, the G-quadruplex ligand is TMPyP4.

[0013] Furthermore, the molar ratio of the G-quadruplex ligand TMPyP4 and entecavir is (0.2-1):1;

[0014] Specifically, the molar ratio of the G-quadruplex ligand TMPyP4 and entecavir is 1:1.

[0015] In a third aspect, the present invention provides a pharmaceutical preparation for preventing HBV infection, which is prepared by using any of the above-mentioned pharmaceutical compositions as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0016] In a fourth aspect, the present invention provides a combination drug for anti-HBV infection, comprising a G-quadruplex ligand and entecavir of the same or different specifications, administered simultaneously or separately, and a pharmaceutically acceptable carrier; the G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

[0017] Compared with the prior art, the advantages of the present invention are as follows: the present invention selects a G quadruplex ligand targeting HBV G quadruplex and entecavir (ETV) for combined use, which can inhibit the production of HBV-related viral proteins and control the pathogenicity of HBV in cells; compared with the use of HBV G quadruplex ligand alone or entecavir alone, the combined use of the two has a stronger therapeutic effect against HBV infection.

[0018] The research results of the present invention can provide new tools and ideas for the prevention and treatment of clinical hepatitis B, especially in the development and clinical application of anti-HBV drugs, and can also be directly applied to scientific research or guide the development of inhibitors that inhibit the HBV virus life cycle, and have important theoretical significance for finding new drug targets and screening new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The sequence information, position diagram and sequence information of 6 potential HBV G-quadruplexes are shown in Figure 2. These sequence information was obtained by analyzing the HBV DNA whole genome sequence through 4 online analysis websites G4CatchAll (homes.ieu.edu.tr / odoluca / G4Catchall / ), ImGQfinder (ImGQfinder(niifhm.ru)), Pqsfinder (pqsfinder–imperfection-tolerant G-quadruplex prediction(muni.cz)) and QGRS (QGRS Mapper | Analyze and Search(ramapo.edu)), as well as 1 bioinformatics analysis software MEGA6. The sequence information of all different subtypes came from the HBV database HBVdb (HBVdb::Home - Hepatitis B Virus Database(inserm.fr)).

[0020] Figure 2 The chemical structures of the G-quadruplex ligands TMPyP4, BRACO19, PDS, and PDP. In the figure, TMPyP2 is a negative control of TMPyP4. The two have similar structures, but TMPyP2 does not bind to the G-quadruplex.

[0021] Figure 3Schematic diagram of the formation of G4 structure by six G-quadruplex sequences verified by CD spectroscopy.

[0022] Figure 4 These are the results of cytotoxicity analysis of the ligands TMPyP4, BRACO19, PDS, and PDP.

[0023] Figure 5-6 These are the validation results of the therapeutic effects of ligands TMPyP4, BRACO19, PDS and PDP on HBV (HBsAg and HBeAg) infection.

[0024] Figure 7 These are the verification results of the inhibitory effects of ligands TMPyP4, BRACO19, PDS and PDP on the toxicity of AD38 cells.

[0025] Figure 8-10 These are the validation results of the combined treatment of ligand TMPyP4 and ETV in reducing the levels of HBV (HBsAg and HBeAg) viral proteins during HBV infection with NTCP. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The present invention found through analysis that there is a conservative G-quadruplex sequence in the HBV DNA genome. The sequence is first synthesized, then heated at 95°C for 5 min under the action of 100mM K ions and slowly annealed to room temperature to form a G4 structure. Finally, it is found through circular dichroism that it can form G4-specific peaks at 265nm and 295nm. The full-length genome sequence of HBV virus is shown in SEQ ID NO.1. The six G-quadruplex sequences are shown in Figure 1 As shown, it is a special region in the full-length genome of HBV virus that can bind to G4 ligand. This shows that all six G-quadruplex sequences can form a stable G4 structure.

[0028] Example 1: CD determination of melting curves to verify the stabilizing effect of ligands

[0029] Six HBV DNA G4 sequences were artificially chemically synthesized and preheated at 95°C for 5 min to form a G4 structure. The operating temperature of the circular dichroism spectrometer was set to 25°C, the scanning wavelength was 220-320 nm, the optical path was 1.0 mm, and the scanning speed was 50 nm / min. The mixed solution was then aspirated into a 1.00 mm cuvette to measure the CD spectra of different G4s sequences. According to the CD spectrum, the wavelength that can achieve maximum light absorption was selected for Tm value detection, and the temperature range was set to 4-95°C. The heating rate was 1°C / min. The final raw data was processed by Origin Pro 8.0 software to draw the final spectrum, as shown below. Figure 3 shown.

[0030] Example 2: Cytotoxicity studies of ligands TMPyP4, BRACO19, PDS and PDP

[0031] The chemical structures of ligands TMPyP4, TMPyP2, BRACO19, PDS and PDP are shown in Figure 2 TMPyP2 is a negative control of TMPyP4. The two have similar structures, but TMPyP2 does not bind to G-quadruplexes.

[0032] HepG2 cells in good growth state were plated into 96-well plates. After culturing for 24 h, different concentration gradients (3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of ligands TMPyP4, BRACO19, PDS and PDP were added. After the culture plates were cultured for another 24 h, 10 μL of CCK8 solution was added to each well. The culture plates were cultured in the incubator for another 2 h, and the absorbance at 450 nm was measured with a microplate reader. The cell viability was calculated after subtracting the background value of the blank well control from the OD.

[0033] The results are as follows Figure 4 As shown, it can be seen that TMPyP4 and PDS have lower cytotoxicity, while PDP has higher cytotoxicity. At high concentrations, it is not suitable for the treatment of HBV infection. BRACO19 has certain cytotoxicity, and the dosage concentration should not exceed 25 μM.

[0034] Example 3: Detection of the inhibitory effect of ligands TMPyP4, BRACO19, PDS, and PDP alone on viral levels in NTCP cells infected with HBV

[0035] After AD38 cells (human hepatoma cell line, provided by Xia Yuchen's laboratory, School of Basic Medical Sciences, Wuhan University; preserved and passaged by this laboratory, and can produce HBV virus) were used to produce virus, the cell culture supernatant was collected, the supernatant was concentrated by centrifugation using a 100kDa ultrafiltration tube, and then sterilized by filtration using a 0.22 μM filter. NTCP cells were plated in a well plate pre-coated with collagen, and when the cells grew to 60%-80%, they were cultured in 2% DMSO medium for 24 h; then the concentrated and sterilized HBV virus solution was added to infect NTCP cells at MOI=200, and the culture supernatant containing HBV was discarded after 24 h; PBS was washed three times, and culture medium containing different concentrations (2 μM, 5 μM and 10 μM) of ligands TMPyP4, BRACO19, PDS and PDP was added, and the culture supernatant was collected after continuing to culture for 48 h. The changes in HBsAg and HBeAg protein levels were measured using an ELISA kit.

[0036] The results are as follows Figure 5 and Figure 6 As shown, compared with the groups without drug addition (Mock) and negative control (TMPyP2), the levels of HBsAg and HBeAg in the supernatant of the culture medium of NTCP cells infected with HBV were significantly reduced after the addition of ligands TMPyP4 and BRACO19, and the levels were concentration-dependent. This was statistically significant.

[0037] Example 4: Inhibitory effects of ligands TMPyP4 and BRACO19 on AD38 cell toxin production

[0038] The AD38 cell line selected in the present invention is within the non-cytotoxic concentration range verified by the CCK8 experiment and can be used for research on drug inhibition of viral expression levels.

[0039] Rat tail type I collagen was used at 5 μg / cm 2 The 12-well plate was coated with the working concentration of , and the collagen solution was discarded after the 12-well plate was placed at room temperature for 1 h. The 12-well plate was washed 3 times with PBS. AD38 cells were added to the coated well plate, and AD38 cells were cultured with DMEM medium (containing 10% FBS and 1% PS) until the density reached 90%. The toxin-producing medium (containing 5% FBS, 1% PS, 1.8% DMSO and 5μg / mL hydrocortisone) was replaced, and different concentrations (2 μM, 5 μM and 10 μM) of ligands TMPyP4 and BRACO19 were diluted in toxin-producing medium in advance. After continuing to culture for 48 h, the medium was discarded, 200 μL of SDS loading was added to each well and heated at 100℃ for 10 min, followed by Western Blot.

[0040] The results are as follows Figure 7As shown, compared with the control group without ligand, the addition of ligands TMPyP4 and BRACO19 can significantly reduce the HBc protein level in a concentration-dependent manner, indicating that the ligands TMPyP4 and BRACO19 can inhibit the translation of HBV proteins.

[0041] Example 5: Detection of the inhibitory effect of the combination of ligand TMPyP4 and entecavir (ETV) on the viral level in NTCP cells infected with HBV

[0042] After AD38 cells were used to produce virus, the cell culture supernatant was collected, the supernatant was concentrated by centrifugation using a 100kDa ultrafiltration tube, and then sterilized by filtration using a 0.22 μM filter. NTCP cells were plated on a well plate pre-coated with collagen, and when the cells grew to 60%-80%, they were cultured for 24 h using a 2% DMSO medium; then concentrated and sterilized HBV virus solution was added to infect NTCP cells at an MOI of 200, and the culture supernatant containing HBV was discarded after 24 h; the cells were washed three times with PBS, and culture medium containing different concentrations (2 μM, 5 μM, and 10 μM) of ligand TMPyP4 or ETV (entecavir, concentration 10 μM) was added alone, or different concentrations (2 μM, 5 μM, and 10 μM) of ligand TMPyP4 and ETV (entecavir, concentration 10 μM) were added at the same time, and the culture supernatant was collected after 48 h of continuous culture. ELISA kits were used to measure changes in HBsAg and HBeAg protein levels in the culture medium supernatant, and antibodies were used to detect changes in intracellular HBsAg and HBxAg protein levels.

[0043] The results are as follows Figure 8 and Fig. 9 As shown, the levels of HBsAg and HBeAg in the supernatant of the culture medium of NTCP cells infected with HBV after the addition of ligands TMPyP4 and ETV were significantly reduced in a concentration-dependent manner compared with the groups without drug addition (AD38, "Mock") and negative control addition (TMPyP2).

[0044] like Figure 8-10 As shown in the figure, compared with the addition of TMPyP4 or ETV alone, the combination of TMPyP4 and ETV can significantly increase the therapeutic effect. When both TMPyP4 and ETV were 10 μM, the therapeutic effect of ETV alone was poor. This shows that the combination of drugs can significantly reduce the changes in HBsAg and HBxAg protein levels in cells, and has a significant effect on improving the efficacy.

[0045] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for preparing a drug for preventing HBV infection by combining a G-quadruplex ligand with entecavir, characterized in that: The G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

2. The application according to claim 1, characterized in that: The G-quadruplex ligand is TMPyP4.

3. The application according to claim 1, characterized in that: The molar ratio of G-quadruplex ligand TMPyP4 and entecavir is (0.2-1):

1.

4. The application according to claim 3, characterized in that: The molar ratio of G-quadruplex ligand TMPyP4 and entecavir was 1:

1.

5. A pharmaceutical composition for resisting HBV infection, characterized in that: It is composed of a G-quadruplex ligand and entecavir, wherein the G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

6. The pharmaceutical composition for resisting HBV infection according to claim 5, characterized in that: The G-quadruplex ligand is TMPyP4.

7. The pharmaceutical composition for resisting HBV infection according to claim 5, characterized in that: The molar ratio of G-quadruplex ligand TMPyP4 and entecavir is (0.2-1):

1.

8. The pharmaceutical composition for resisting HBV infection according to claim 7, characterized in that: The molar ratio of G-quadruplex ligand TMPyP4 and entecavir was 1:

1.

9. A pharmaceutical preparation for resisting HBV infection, characterized in that: The pharmaceutical composition is prepared by taking the pharmaceutical composition according to any one of claims 5 to 8 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

10. A combined drug for preventing HBV infection, characterized in that: Contains a G-quadruplex ligand and entecavir of the same or different specifications, which are administered simultaneously or separately, and a pharmaceutically acceptable carrier; the G-quadruplex ligand is any one or more of TMPyP4, BRACO19, PDS, and PDP.

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