CN110812472B

CN110812472B - Application of E3 ubiquitin ligase stub1 in inhibiting replication of hepatitis B virus

Info

Publication number: CN110812472B
Application number: CN201911144836.8A
Authority: CN
Inventors: 林旭; 林嘉成
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2022-10-21
Anticipated expiration: 2039-11-19
Also published as: CN110812472A

Abstract

The invention discloses an application of E3 ubiquitin ligase stub1 in inhibiting hepatitis B virus replication, relates to the field of biomedicine, and relates to an application of an E3 ubiquitin ligase stub1 gene or an expression product of the E3 ubiquitin ligase stub1 gene in inhibiting hepatitis B virus replication. The E3 ubiquitin ligase stub1 provided by the invention can ubiquitinate a substrate protein of HSP90, is combined with hepatitis B virus core protein HBc, shortens the half-life period of the substrate protein, down-regulates capsid protein, effectively inhibits the replication of hepatitis B virus, overcomes the defects of fast metabolism and short half-life period of capsid protein inhibitors such as HAPs and other drugs, and has good application prospect in the aspects of inhibiting and eliminating hepatitis B virus. Furthermore, since E3 ubiquitin ligase stub1 needs to form an adapter with HSP70, HSP90 and degrade capsid protein of hepatitis b, HSP90 inhibitor can enhance its function, exerting similar function to stub 1.

Description

Application of E3 ubiquitin ligase stub1 in inhibiting replication of hepatitis B virus

Technical Field

The invention relates to the field of biological medicine, in particular to application of E3 ubiquitin ligase stub1 in inhibiting replication of hepatitis B virus.

Background

Hepatitis B Virus (HBV) belongs to the hepadnaviridae family. HBV infection causes chronic hepatitis B and is closely related to the occurrence and development of cirrhosis and liver cancer. More than 50% of primary liver cancer is related to HBV. China is a high-incidence area of HBV infection, and more than one hundred million people of HBV carriers exist. The currently approved antiviral drugs are mainly interferon (including IFN-alpha and PEG-IFN) and nucleotide analogs, which can inhibit virus reverse transcriptase and reduce virus titer, but have low clearance rate for hepatitis B virus.

HBV core protein (HBc), also known as capsid protein, is a viral structural protein and is the major constituent of HBV nucleocapsid. Inhibitors against capsid proteins are currently a hot spot in the development of anti-HBV drugs. Deres, K et al, in screening for agents that inhibit hepatitis B replication, found that Heteroaryldihydropyrimides (HAPs), such as Bay41-4109, inhibited normal assembly of the nucleocapsid by binding to HBc, while allowing the half-life of HBc to be greatly shortened. At present, some HAPs compounds are in phase I and phase II clinical experiments, but the metabolism speed of the compounds in vivo is fast, and the effect at an animal level is still to be improved.

Replication of HBV requires the help of many cellular proteins such as heat shock protein 90 (HSP 90) and the like. Meanwhile, some cellular proteins such as apobec3c and the like are also limiting factors for replication of HBV. Stub1 is an E3 ubiquitin ligase that ubiquitinates the substrate protein that binds to heat shock proteins. Whether Stub1 ubiquitinates the substrate protein of HSP90 determines whether the substrate protein is refolded by HSP90 or degraded. Thus, stub1 plays a key role in protein quality control and protein balance of cells.

Therefore, those skilled in the art are devoted to develop an E3 ubiquitin ligase which can down-regulate hepatitis B virus capsid protein, make up for the defects of fast metabolism and short half-life of HAPs and other drugs, and improve the effect of inhibiting hepatitis B virus replication.

Disclosure of Invention

In view of the above defects in the prior art, the technical problem to be solved by the present invention is how to develop an E3 ubiquitin ligase, which has the effects of down-regulating hepatitis b virus capsid protein and efficiently inhibiting replication of hepatitis b virus, and does not have the disadvantages of fast metabolism, short half-life period, etc.

In order to achieve the purpose, the invention provides an application of E3 ubiquitin ligase stub1 in inhibiting replication of hepatitis B virus.

Furthermore, the application refers to the application of the E3 ubiquitin ligase stub1 gene or the expression product of the E3 ubiquitin ligase stub1 gene in inhibiting the replication of the hepatitis B virus.

Further, the expression product of the E3 ubiquitin ligase stub1 gene includes the stub1 protein, and a mutant and a functional domain of stub1 having the activity.

Further, the E3 ubiquitin ligase stub1 gene can play a role in delivering to a cell infected with hepatitis b virus through a vector.

Further, the vector comprises adenovirus, adeno-associated virus, lentivirus and plasmid.

Further, the E3 ubiquitin ligase stub1 can bind to the client protein HBc of HSP90, shorten its half-life, and thereby down-regulate HBc protein levels.

Further, it can also be used in combination with HSP90 inhibitors by enhancing the function of said E3 ubiquitin ligase stub 1.

Further, the HSP90 inhibitor includes geldanamycin and derivatives thereof.

Further, it can be used in combination with capsid protein inhibitor.

Further, the capsid protein inhibitors include isoaryldihydropyrimidine HAPs.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) The E3 ubiquitin ligase stub1 provided by the invention can be efficiently combined with core protein HBc and promote the degradation of the core protein HBc, so that hepatitis B virus capsid protein is remarkably reduced, and the replication of hepatitis B virus is inhibited;

(2) The E3 ubiquitin ligase stub1 provided by the invention can make up the defect that capsid protein inhibitors such as HAPs and other drugs are metabolized faster, and improve the inhibition efficiency.

The conception, specific structure and technical effects of the present invention will be further described in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

Drawings

FIG. 1 is a diagram showing that stub1 reduces the level of HBV viral DNA secreted from HepAD38 cells according to a preferred embodiment of the present invention;

FIG. 2 is a diagram showing that stub1 according to a preferred embodiment of the present invention reduces the level of hepatitis B virus core protein in HepGAD38 cells;

FIG. 3 is a schematic diagram showing the effect of stub1 in regulating the half-life of HBc according to a preferred embodiment of the present invention;

FIG. 4 is a schematic diagram illustrating the effect of an Hsp90 inhibitor in regulating the half-life of HBc according to a preferred embodiment of the present invention;

FIG. 5 is a graph showing the results of stub1 increasing the level of ability of Bay41-4109 to inhibit hepatitis B virus secretion from HepAD38 cells according to a preferred embodiment of the present invention;

FIG. 6 is a graph showing the results of the level of inhibition of the ability of the HepAD38 cells to secrete hepatitis B virus by increasing the level of Bay41-4109 inhibition by an inhibitor of Hsp90 according to a preferred embodiment of the present invention;

FIG. 7 is a schematic diagram showing the results of stub1 down-regulating HBV DNA levels in serum of HBV transgenic mice according to a preferred embodiment of the present invention;

FIG. 8 is a schematic representation of the drug processing flow of caudal vein injection of adeno-associated virus type 8 to overexpress stub1 and Bay41-4109 and 17AAG in the liver tissue of transgenic mice according to a preferred embodiment of the invention;

FIG. 9 is a schematic diagram showing the inhibitory effect of stub1 or 17AAG on the serum HBV DNA of HBV transgenic mice to increase Bay41-4109, according to a preferred embodiment of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 effects of stub1 and HSP90 inhibitors on inhibition of HBV in cells and in combination with HAPs

We used the HepAD38 cell line integrated with HBV genome, which was able to stably produce infectious HBV particles without tetracycline induction. Recombinant adenovirus (Ad-stub 1) for expressing stub1 or empty vector control virus (Ad-GFP) is used for infecting HepAD38 cells, culture solution is replaced for the cells while infection is carried out, and the culture solution is taken out after 3 days and is detected by a hepatitis B virus DNA detection kit (Shengxiang).

As a result, as shown in FIG. 1, extracellular viral DNA was decreased when stub1 was overexpressed. HepAD38 cells were lysed and the HBc and actin proteins were detected using a western blot (as shown in FIG. 2). As a result, it was found that overexpression of stub1 significantly reduced the HBc protein level, while the empty vector did not cause any difference.

To investigate whether stub1 could reduce the half-life of HBc, we blocked protein synthesis in Huh7-HBc cells by Cycloheximide (CHX) and followed HBc protein levels within 24 hours. Although HBc is very stable, when stub1 is overexpressed, the half-life of HBc is shortened from 19 hours to around 6 hours in Huh7-HBc cells (as shown in fig. 3). The inhibitor of Hsp90, 17AAG, also significantly reduced the half-life of HBc by treating the cells (as shown in figure 4).

Since overexpression of stub1 has an effect of shortening half-life of HBc, we tested the inhibitory effect of Bay41 after overexpression of stub 1. HepAD38 cells were treated with 0-8. Mu.M Bay41-4109, infected 2 days later with Ad-stub1 or control Ad-GFP, and 4 days later with culture broth collected for HBV DNA detection. As a result, stub1 significantly improved Bay41 efficacy (as shown in fig. 5). Likewise, the inhibitor of HSP90, 17AAG, also significantly increased the efficacy of Bay41-4109 (as shown in figure 6).

Example 2 effects of stub1 and HSP90 inhibitors on HBV inhibition and combination with HAPs in hepatitis B transgenic mice

Selecting C57BL/6 type HBV transgenic male mice with the age of 8 weeks, 5 experimental groups and 5 control groups, wherein the HBV virus titer in the serum of the selected mice is about 5-6 multiplied by 10 ⁶ IU/ml. stub1 experimental group recombinant adeno-associated virus type 8 expressing stub1 (AAV-stub 1) was injected intravenously into the tail of each mouse at 1x10 ¹² v.g/mouse, each mouse was injected tail vein with the same dose of empty vector control virus (AAV-GFP) 20 days after injection as day 0. Orbital bleeds were performed on

days

0, 6, 12, 18, 24 and 30, and sera were taken to measure virus titers in mouse sera.

As shown in FIG. 7, the HBV DNA content in the serum of the control mice was 5.0 to 8.0X 10 in the first 20 days ⁶ IU/ml. The HBV DNA content in the serum of the experimental group mice is generally reduced from 6.0 multiplied by 10 ⁶ IU/ml is reduced to 1.5X 10 ⁶ IU/ml. This indicates that stub1 reduces viral titers in serum of HBV transgenic mice.

We also investigated whether stub1 and Hsp90 inhibitor 17AAG increased the inhibitory effect of Bay41-4109 on HBV in HBV transgenic mice. Mice were divided into 4 different experimental groups (5 per group) and treated with i) control solvent and AAV-GFP, ii) Bay41-4109 and AAV-GFP, iii) Bay41-4109 and AAV-stub1, iv) Bay41-4109 and 17AAG. Administration and AAV treatment time AAV was administered three weeks in advance by tail vein injection, bay41-4109 (10 mg/kg body weight) and 17AAG (50 mg/kg body weight) intraperitoneally at twice a day and once every two days, respectively, as shown in FIG. 8. Bay41-4109 inhibited serum HBV DNA by 1.8log 10copies/ml on day 30 of administration. BAY41-4109 in combination with 17AAG or stub1 overexpression further inhibited about 1log 10copies/ml in serum (as shown in FIG. 9).

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. An application of E3 ubiquitin ligase stub1 in preparing a medicament for inhibiting hepatitis B virus.

2. The use of the E3 ubiquitin ligase stub1 in the preparation of a medicament for inhibiting hepatitis b virus according to claim 1, which is characterized in that the use of the E3 ubiquitin ligase stub1 gene or the expression product of the E3 ubiquitin ligase stub1 gene in inhibiting hepatitis b virus replication.

3. The use of E3 ubiquitin ligase stub1 in the preparation of a medicament for inhibiting hepatitis b virus according to claim 2, wherein the expression product of E3 ubiquitin ligase stub1 gene comprises stub1 protein, and mutants and functional domains of stub1 having the same activity as the stub1 protein.

4. The use of the E3 ubiquitin ligase stub1 of claim 2 in the preparation of a medicament for inhibiting hepatitis b virus, wherein the E3 ubiquitin ligase stub1 gene is delivered into a cell infected with hepatitis b virus by a vector to function.

5. The use of E3 ubiquitin ligase stub1 in the preparation of a medicament for inhibiting hepatitis B virus according to claim 4, wherein the vector comprises adenovirus, adeno-associated virus, lentivirus, plasmid.

6. The use of the E3 ubiquitin ligase stub1 in the preparation of a medicament for inhibiting hepatitis b virus according to claim 1, wherein the E3 ubiquitin ligase stub1 is capable of binding to the client protein HBc of HSP90, shortening its half-life and thereby down-regulating HBc protein levels.

7. The use of E3 ubiquitin ligase stub1 in the manufacture of a medicament for inhibiting hepatitis b virus according to claim 1, wherein the effect of inhibiting replication of said hepatitis b virus is enhanced in combination with an HSP90 inhibitor.

8. The use of E3 ubiquitin ligase stub1 in the manufacture of a medicament for the inhibition of hepatitis b virus according to claim 7 wherein said HSP90 inhibitor comprises geldanamycin or a derivative thereof.

9. The use of the E3 ubiquitin ligase stub1 in the preparation of a medicament for inhibiting hepatitis b virus according to claim 1, wherein the E3 ubiquitin ligase stub1 is used in combination with a capsid protein inhibitor.

10. The use of E3 ubiquitin ligase stub1 in the manufacture of a medicament for inhibiting hepatitis b virus according to claim 9 wherein the capsid protein inhibitors comprise heteroaryl dihydropyrimidine HAPs.