CN110251495B

CN110251495B - Application of 20-deoxyingenol in preparation of medicine for inhibiting HBV replication

Info

Publication number: CN110251495B
Application number: CN201810198418.6A
Authority: CN
Inventors: 胡接力; 黄爱龙; 魏霞飞
Original assignee: Chongqing Medical University
Current assignee: Chongqing Mingdao jietest Biotechnology Co.,Ltd.
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2022-05-31
Anticipated expiration: 2038-03-12
Also published as: CN110251495A

Abstract

The invention discloses an application of 20-deoxyingenol in preparing a medicament for inhibiting HBV replication. According to the invention, the cell model is used as a screening tool, and the screened natural compound 20-deoxyingenol is proved to have good activity of inhibiting HBV replication in cells through research, and further research on an action mechanism shows that the action mechanism influences HBC dimer formation, thereby influencing nucleocapsid formation; the 20-deoxyingenol can be used for research of hepatitis B treatment to develop a novel anti-HBV treatment drug taking the nucleocapsid as a target spot, and the drug targeting the nucleocapsid can effectively inhibit virus replication and is different from the existing nucleoside drug in pharmacology, so that the drug can be used together with the nucleoside drug or can be used for alternative treatment after the nucleoside drug is resistant, and the drug resistance problem of the existing drug is relieved.

Description

Application of 20-deoxyingenol in preparation of medicine for inhibiting HBV replication

Technical Field

The invention belongs to the field of molecular biology, and relates to application of a natural compound in inhibition of HBV replication, in particular to application of 20-deoxyingenol in a medicine for inhibiting HBV replication.

Background

Hepatitis B Virus (HBV) infection is a serious public health problem involving a wide range of nearly 4 billion chronically infected patients worldwide, and chronic HBV infection can lead to severe consequences such as cirrhosis, hepatocellular carcinoma, and so on, with nearly a million deaths each year.

At present, the drugs for treating hepatitis B mainly comprise alpha interferon (IFN-alpha, PEG-IFN alpha) and 6 nucleoside (acid) analogues (lamivudine, adefovir, entecavir, telbivudine, tenofovir alafenamide). After 48 weeks of long-acting interferon (PEG-IFN α) treatment, only a few patients achieved sustained responses with significant side effects. Nucleoside (acid) analogues exhibit strong viral inhibitory effects in most patients, and they can reduce the serum HBV load of most patients over a period of time, improve transaminase levels and liver histological manifestations. However, there are some problems associated with the treatment with nucleoside (acid) drugs, including rebound after withdrawal of the drug, and drug resistance caused by screening of drug-resistant mutants with long-term drug administration. In conclusion, the existing therapeutic drugs are far from enough to completely solve the problem of hepatitis B, and the development of novel anti-HBV drugs becomes urgent in the field of hepatitis B research at present.

The development of novel anti-HBV drugs aims at novel targets, and the nucleocapsid is an ideal anti-viral target. Hepatitis B virus has multiple components, and effective medicaments should be directed against the factors which are crucial in the hepatitis B virus. In addition to HBV DNA polymerase, the nucleocapsid composed of the core protein is another key component in the replication process of HBV. The nucleocapsid serves on the one hand as an important structure of HBV, packaging HBV pregenomic RNA (pgRNA), containing genomic DNA, and on the other hand also plays an important functional role in the replication of viral DNA. Nucleocapsid loss, or functional defect, can result in failure to carry out or severely impaired viral replication. Therefore, the development of new anti-HBV therapeutic drugs, the nucleocapsid, is a good candidate target, because the drug targeting the nucleocapsid can effectively inhibit virus replication and is different from the existing nucleoside drugs in pharmacology, so that the drug can be used in combination with the nucleoside drugs or can be used for alternative treatment after the nucleoside drugs are resistant.

The HBV nucleocapsid is composed of core protein (HBC). The basic unit of the nucleocapsid is the dimer of HBC, and one nucleocapsid contains 90 or 120 dimers of HBC. Nucleocapsids are generally formed in at least two steps, first by dimer formation from HBC monomers and second by polymerization between dimers to form multimers and even intact nucleocapsids. It is presently believed that the process from dimer to nucleocapsid first undergoes a relatively slow trimerization step, called nucleation, followed by a rapidly extending phase centered on the hexamer, which rapidly aggregates to form the nucleocapsid.

From the above description, it can be seen that, regardless of the specific process, the formation of a nucleocapsid involves two different interactions, one of which is the interaction between the monomers of the core protein and the other of which is the interaction between the two dimers. Accordingly, observation of the HBC monomer from the nucleocapsid structure reveals at least two interacting interfaces, the first located within the dimer and the other located between two adjacent dimers. From the point of view of drug development, interfering with nucleocapsid formation can be initiated by interfering with the interaction between the monomers of the core protein and thus with dimer formation, and by interfering with the interaction between dimers and thus with the multimerization process of the dimers.

To date, no nucleocapsid targeting drugs are available on the market, but five classes of compounds have potential pharmaceutical value, namely, Heteroaryldioxidines (HAPs), represented by BAY41-4109, GLS 4; phenylallylacrylamide derivatives (PPAs), represented by AT130 and AT 61; sulfonamide Benzamide derivatives (Sulfamoylbenzamide, SBA) or formamide derivatives (Benzamide, BA); isothiafludine (Isothiafludine, NZ-4); pyridazinone derivatives (pyridazinones), represented by 3771. The five compounds achieve the effect of inhibiting HBV replication by influencing the formation or function of nucleocapsid. Their target is the capsid assembly process, the mechanism is to promote the misassembly of nucleocapsids, or to prevent nucleocapsids from normally packaging pgRNA, the known site of action is the site between two dimers, and there is no evidence to show that these compounds interfere with HBC dimer formation.

In order to screen compounds targeting HBC dimer formation, the applicant previously constructed a drug screening cell model cell line NCTP6 targeting hepatitis B virus core protein dimer formation (see Chinese patent application 201611166574.1).

Disclosure of Invention

The invention aims to provide application of 20-deoxyingenol in a medicine for inhibiting HBV replication.

The application is the application of 20-deoxyingenol in preparing a medicine for inhibiting HBV DNA replication.

In the above technical scheme, the application is the application of 20-deoxyingenol in preparing a medicament for promoting HBC dimer and nucleocapsid formation.

The invention has the beneficial effects that: the natural compound 20-deoxyingenol screened from a compound library containing 672 synthetic compounds and natural compounds by taking a cell model constructed in the previous patent application of the applicant as a screening tool is proved to have good activity of inhibiting HBV replication in cells, and further research on an action mechanism shows that the action mechanism influences HBC dimer formation so as to influence the formation of nucleocapsid; the 20-deoxyingenol can be used for research of hepatitis B treatment to develop a novel anti-HBV treatment drug taking the nucleocapsid as a target spot, and the drug targeting the nucleocapsid can effectively inhibit virus replication and is different from the existing nucleoside drug in pharmacology, so that the drug can be used together with the nucleoside drug or can be used for alternative treatment after the nucleoside drug is resistant, and the drug resistance problem of the existing drug is relieved.

Drawings

FIG. 1 is a flow chart of a preliminary screening of compounds using the cell line NCTP 6.

FIG. 2 shows the results of the first round of screening of the batch in which 20-deoxyingenol was present when screening was performed using the cell line NCTP 6.

FIG. 3 shows the dose-response relationship of 20-deoxyingenol to the activity of Rluc of NCTP6 cells.

FIG. 4 is a graph showing the results of the test of the effect of 20-deoxyingenol on the activity of RlucN-L-C, wherein A: a schematic of the structure of plasmid pRlucN-L-C; b: results of the effect of various concentrations of 20-deoxyingenol treatment on luciferase activity of RlucN-L-C.

FIG. 5 is an experimental validation of 20-deoxyingenol-facilitated nucleocapsid formation, wherein A: 3flag-HBC can support verification results of nucleocapsid formation; b: the core capsid formed by 3flag-HBC can support the experimental result of HBV DNA replication; c: 3flag-HBC can support the experimental result of the formation of HBV relaxed circular DNA (rcDNA); d: experimental results of the 20-deoxyingenol facilitated the formation of dimers and nucleocapsids from 3 flag-HBC.

FIG. 6 shows the results of experiments on the promotion of nucleocapsid formation in HepAD38 cells by 20-deoxyingenol, where WB: HBC is HBC expression detected by Western blot, and β -Actin is internal control.

FIG. 7 shows the result of HBV coreDNA Southern blot assay in HepAD38 and HepG2.2.15 cells after 20-deoxyingenol treatment.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Sources of reagent materials used:

cell lysis solution: the formula is as follows: 1ml of 1M Tris-HCl (PH 8), 200. mu.l of 0.5M EDTA, 40200. mu.l of NP, and adding distilled water to the solution to make the volume of the solution constant to 100 ml;

digoxin probe labeling and Southern blot detection kit: roche, germany;

rluc luciferase assay kit: promega corporation, usa;

lipofectamine 3000 transfection reagent: thermo Scientific, usa;

anti-Flag monoclonal antibodies: sigma, USA;

anti-GAPDH antibody, anti- β -Actin antibody: bi yun tian, china;

IR Dye-labeled secondary antibody: licor corporation, usa;

natural compound library: medchexpress corporation and seleck corporation, usa;

library of anti-infective synthetic compounds: medchexpress corporation, usa;

HEK293 cells, hepg2.2.15 cells: american mode strain collection center;

HepAD38 cells: constructed by a Robert King laboratory and presented by the Tang Red teaching of the Huaxi hospital of Sichuan university;

cellulose nitrate membrane: pall corporation, usa;

PrimeSTAR Max: takara corporation, japan;

and (3) glue recovery kit: QIAGEN, germany;

BsmB I, Tango buffer, DTT: thermo scientific, usa;

t7 ligase: enzymetics, Inc., USA;

ATP: new England Biolabs, USA.

20-deoxyingenol C20H28O4, available from MCE corporation, usa, having a molecular weight of 332.43 and a molecular structure:

example 1

First, preliminary screening of Compound library

The NCTP6 cell line (see Chinese patent application 201611166574.1) constructed by the applicant at the earlier stage is used as a screening model, two types of compound libraries are preliminarily screened, the first type is synthetic compounds with anti-infection (antibacterial, virus and fungus) activity, and 368 types are totally selected; the other is a total of 304 natural compounds with biological activity. The primary screening procedure for compounds is shown in FIG. 1, where NCTP6 cells were first cultured 24h before inoculation, in tetracycline-free medium; the next day, the NCTP6 cells were seeded in 96-well plates, and 6h after seeding, the compound to be screened was added to each well to a final concentration of 20 μ M (each compound was repeated 3 times); after 48h of dosing, the Rluc luciferase activity of each well was detected. Artificially, compounds that decreased Rluc activity by more than 3-fold, or increased by more than 2-fold, and did not cause significant cell death by microscopic visual observation, when compared to the negative control with DMSO alone, were candidates for the second round of screening.

According to the above criteria, among 304 natural compounds screened, 42 compounds capable of reducing the Rluc activity by 3-fold or more, 6 compounds capable of increasing the Rluc activity by 2-fold or more, and 36 compounds in total, except those capable of causing significant cell death, were allowed to enter the next round of screening as candidate compounds. In FIG. 2, the results of the first round of screening of the screening lot in which 20-deoxyingenol was present are shown, and it can be seen that 20-deoxyingenol increased the Rluc activity in NCTP6 cells by more than 2-fold at a concentration of 20. mu.M.

Di, 20-deoxyingenol has dose-dependent enhancing effect on Rluc activity of NCTP6

To further confirm the effect of 20-deoxyingenol on the Rluc activity of NCTP6 cells, cells were treated at 9 final concentrations, including 0 μ M, 0.2 μ M, 0.5 μ M, 1 μ M, 2 μ M, 5 μ M, 10 μ M, 20 μ M, 50 μ M. Changes in Rluc activity were measured 48h after compound addition to NCTP6 cell culture medium treatment (3 wells in each concentration). As shown in FIG. 3, the potentiating effect of 20-deoxyingenol on Rluc activity showed a dose-dependent effect. Meanwhile, toxicity tests showed that 20-deoxyingenol has CC50> 100. mu.M on NCTP6 cells.

The tri, 20-deoxyingenol does not affect the activity of the Rluc luciferase

The potentiation of the Rluc activity of NCTP6 cells by deoxyingenol suggests that this compound may influence the formation of HBC dimers, but the possibility of: i.e. the enhancement of Rluc activity we have seen, results from the fact that this compound directly affects the fragmented Rluc, thus enhancing its activity. To rule out this possibility, we constructed a plasmid pRlucN-L-C that expresses a RlucN linked to RlucC by a G4S linker (glycine-serine linker of length 47aa, cf. Chinese patent application 201510075723.2) (FIG. 4A). The specific construction process of the plasmid pRlucN-L-C is as follows: plasmid RlucN-HBC (refer to Chinese patent application 201610564291.6) is used as a template, primers Fvect SV40 (5'-ACTCACCGTCTCTTAACTGGCCGCGACTCTAGATCAT-3') and Rg4s GG (5'-TGCGTCCGTCTCTAGATCCACCTCCTCCAGATCCA-3') are used for PCR reaction, and the reaction system is as follows: plasmid RlucN-HBC 1ng, primers Fvect SV40 (10. mu.M) and Rg4s GG (10. mu.M) were each 1. mu.l, PrimeSTAR Max 25. mu.l, and a volume of sterilized ultrapure water was replenished to 50. mu.l. Reaction conditions are as follows: 10s at 98 ℃, 5s at 58 ℃, 20s at 72 ℃ and 35 cycles. Recovering the amplified fragment with a gel recovery kit, and naming the recovered fragment as frag 1; plasmid RlucC-HBC (refer to patent application 201610564291.6) is used as a template, primers Frluc C229GG2 (5'-ACTCACCGTCTCTATCTTCAGGCAAATCTGGTAATGGTTC-3') and Rrluc C229GG2 (5'-GCTGACCGTCTCAGTTATTGTTCATTTTTGAGAACTCGCT-3') are used for PCR reaction, and the reaction system is as follows: plasmid RlucC-HBC 1ng, primers Frluc C229GG2 (10. mu.M) and Rrluc C229GG2 (10. mu.M) were each 1. mu.l, PrimeSTAR Max 25. mu.l, and a volume of sterilized ultrapure water was made up to 50. mu.l. Reaction conditions are as follows: 10s at 98 ℃, 5s at 58 ℃, 5s at 72 ℃ and 35 cycles. The amplified fragment was recovered using a gel recovery kit and the resulting fragment was named frag 2. Then, performing Golden gate connection on the two fragments, wherein the reaction system is as follows:

BsmR I enzyme	0.75μl
		Tango buffer	1μl
DTT	1μl
		T7 ligase	0.25μl
ATP	1μl
		frag1	2μl(80ng)
frag2	2μl(80ng)
		ddH₂O	Make up to 10 μ l
Total volume	10μl

Reaction conditions are as follows: circulating for 25 times at 37 deg.C for 5min and 20 deg.C for 5 min. The inactivation reaction is carried out at 80 ℃ for 20 min. And transforming and sequencing the reaction product to obtain the plasmid pRlucN-L-C.

After transfection of pRlucN-L-C into HEK293 cells, we treated with different concentrations of 20-deoxyingenol. The results show (as shown in figure 4B) that Rluc activity is not affected by treatment with this compound, suggesting that 20-deoxyingenol does not directly affect the activity of the fragment Rluc.

Tetrahydroingenol, 20-deoxyingenol, promotes nucleocapsid formation

To further verify the effect of 20-deoxyingenol on nucleocapsid formation, we constructed a 3xflag tagged HBC expression plasmid (p3flag-HBC) specifically constructed as follows: plasmid 3xflag-RlucN-C (from Chinese patent application 201610564291.6) is used as a template, primers Rg4s GG and Famp (5'-TGCGTCCGTCTCCTTCGTTCCACTGAGCGTCAGA-3') are used for PCR reaction, and the reaction system is as follows: plasmid 3 xflag-RlucN-C1 ng, primers Rg4s GG (10. mu.M) and Famp (10. mu.M) each 1. mu.l, PrimeSTAR Max 25. mu.l, sterile ultrapure water to make up the volume to 50. mu.l. Reaction conditions are as follows: 10s at 98 ℃, 5s at 55 ℃, 15s at 72 ℃ and 35 cycles. Recovering the amplified fragment with a gel recovery kit, and naming the recovered fragment as frag 3; plasmid 3xflag-RlucN-C is taken as a template, and primer FHBCGG (5'

-GCTGACCGTCTCTATCTGACATCGACCCTTATAAAGAA-3 ') and Ramp (5'-GCTGACCGTCTCTCGAAAACTCACGTTAAGGGAT-3') in a PCR reaction system: plasmid 3 xflag-RlucN-C1 ng, primers FHBG (10. mu.M) and Ramp (10. mu.M) each 1. mu.l, PrimeSTAR Max 25. mu.l, sterile ultrapure water make-up volume to 50. mu.l. Reaction conditions are as follows: 10s at 98 ℃, 5s at 55 ℃, 20s at 72 ℃ and 35 cycles. The amplified fragment was recovered with a gel recovery kit, and the recovered fragment was named frag 4. And then, connecting the two fragments by Golden gate, carrying out conversion and sequencing identification on a reaction product to obtain the plasmid p3flag-HBC, wherein the reaction system and the conditions are the same as those in the previous step.

After the plasmid p3flag-HBC and the plasmid HBV1.1c- (from Chinese patent application 201510075723.2) were co-transfected into HepG2 cells, the expressed fusion protein 3flag-HBC was confirmed to perfectly support nucleocapsid formation (as shown in FIG. 5A) and HBV DNA replication (as shown in FIGS. 5B and 5C), indicating that the 3flag-HBC retained the normal function of HBC.

After transfection of HepG2 cells in 24-well plates with p3flag-HBC, the cells were treated with varying concentrations of 20-deoxyingenol for 4 days and then examined for nucleocapsid formation in the cells. The specific method comprises the following steps: cell lysis is carried out for 10min at room temperature by using 100 mul of lysate, the lysate is transferred into a 1.5ml centrifuge tube, centrifugation is carried out for 5min at13,000 Xg, the supernatant is taken for agarose gel electrophoresis, after the electrophoresis is finished, a sample is transferred onto a nitrocellulose membrane, sealing is carried out after fixation, primary antibody and infrared fluorescence secondary antibody are incubated, and then detection is carried out by using a Licor infrared fluorescence detector. As a result, as shown in FIG. 5D, as the treatment concentration of 20-deoxyingenol increased, the formation amount of nucleocapsid was seen to gradually increase, and the total amount of 3flag-HBC and the amount of dimer showed an increasing tendency.

Penta, 20-deoxyingenol promotes nucleocapsid formation in HepAD38 cells

Next, the effect of 20-deoxyingenol on HBV nucleocapsid formation was further examined on HepAD38 cells. HepAD38 cells seeded in 24-well plates were treated with varying concentrations of 20-deoxyingenol for 6 days, lysed, and the nucleocapsids detected therein. As shown in FIG. 6, with increasing concentrations of 20-deoxyingenol, it was seen that the amount of nucleocapsid and HBC increased gradually.

Hexa, 20-deoxyingenol inhibits replication of HBV DNA

To examine the biological effect of 20-deoxyingenol on HBV replication, HepAD38 and HepG2.2.15 cells inoculated in 12-well plates were treated with different concentrations of 20-deoxyingenol, respectively, and after 6 days, intracellular HBV Core DNA was extracted and examined using Southern blot, and as a result, as shown in FIG. 7, it was seen that as the concentration of 20-deoxyingenol increased, both intracellular Core DNA exhibited dose-dependent decreases with IC50 of 31.2. + -. 3.1. mu.M and 14.5. + -. 1.6. mu.M, respectively.

Claims

Use of 20-deoxyingenol as the sole active ingredient in the manufacture of a medicament for inhibiting HBV replication.
2. The use of claim 1, wherein: the application is the application of 20-deoxyingenol in preparing a medicine for inhibiting HBV DNA replication.
3. Use according to claim 2, characterized in that: the application is the application of 20-deoxyingenol in preparing a medicament for promoting HBC dimer and nucleocapsid formation.