CN115920006A

CN115920006A - Application of ABCF1 or agonist thereof in preparation of anti-DNA virus preparation

Info

Publication number: CN115920006A
Application number: CN202211136878.9A
Authority: CN
Inventors: 马春红; 武专昌; 任彩月; 高立芬; 梁晓红; 孙杨; 李春阳; 岳学田; 张召英; 傅振东; 张言坤
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-04-07
Anticipated expiration: 2042-09-19
Also published as: CN115920006B

Abstract

The invention relates to application of ABCF1 or an agonist thereof in preparation of an anti-DNA virus preparation. The research of the invention verifies that the novel cccDNA interacting protein ABCF1 (ATP-binding cassette sub-family Fmember 1) can be combined with cccDNA, and the cccDNA transcription is inhibited through phase separation, so that the application research of HBV replication is inhibited, the key effect of the combination of the N-terminal domain of the ABCF1 and the cccDNA is further determined, the application of the ABCF1 or the agonist thereof in the research and development of anti-HBV infection related drugs is provided, and the important medical development prospect is achieved.

Description

Application of ABCF1 or agonist thereof in preparation of anti-DNA virus preparation

Technical Field

The invention belongs to the technical field of HBV virus inhibitory active ingredients, and particularly relates to application of ABCF1 or an agonist thereof in preparation of an anti-DNA virus preparation.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Hepatitis B Virus (HBV) is a hepadnavirus, chronic infection of which is closely related to cirrhosis and liver cancer, is an important inducing factor for liver cancer, and seriously threatens human health. HBV replication process is complicated, and viral particles bind to NTCP receptor on the surface of hepatocyte and enter hepatocyte, and its genomic DNA (RC-DNA) enters into nucleus, and forms covalently closed circular DNA (cccDNA) through a series of repair mechanisms. The cccDNA serves as a unique template for virus transcription, combines host cell histones, virus core proteins and the like to form a minichromosome to mediate long-term persistence of the virus, and the interaction between the host protein and the cccDNA is crucial to the formation, stability and transcription activity of the cccDNA. The cccDNA has stable structure and long half-life period, is not easy to be degraded, is a repository of in vivo viruses and is also an important reason for persistent existence and difficult clearance after HBV chronic infection. However, it is not clear how the host protein recognizes cccDNA and regulates HBV replication. Therefore, the deep research on the interacting protein of HBV cccDNA and the molecular mechanism for regulating the activity of the cccDNA has important significance for discovering new HBV replication intervention targets and strategies.

The innate immune cells and liver cells recognize HBV through a Pattern Recognition Receptor (PRR) and induce the production of interferon, which is an important prerequisite for the body to stimulate effective immune response and eliminate HBV. Therefore, the determination of the regulatory factor targeting HBV innate immune response is very important for the prevention and treatment of HBV, and is a hotspot and a difficult problem in the field of hepatitis B virus research at present. Based on the cccDNA-hepatocyte nuclear protein interaction screening model established in our earlier stage, an important innate immune regulatory factor ABCF1 was identified in this study. ABCF1 was first discovered in 1998 as a TNF α stimulating gene, a member of the ABCF subfamily of the ATP-binding cassette (ABC) transporter superfamily. Unlike other subfamily members, ABCF1 lacks a transmembrane domain and is expressed in the nuclear cytoplasm. ABCF1 consists of 804 amino acids and comprises an N-terminal domain (ND) and 2 nucleic acid binding domains (NBD). Research shows that ABCF1 can interact with ISD to regulate interferon signal path and play important role in identifying natural immune response of DNA. In addition, there are studies showing that ABCF1 is identified as a co-activator of OCT4 and SOX2, activating pluripotency genes by phase separation, is important for self-renewal of stem cells. However, whether ABCF1 recognizes cccDNA and regulates HBV replication has not been reported so far.

Disclosure of Invention

Aiming at the research background, the invention defines that the ABCF1 as the interacting protein of cccDNA can participate in the regulation of interferon response, and inhibits the transcription of cccDNA and the replication of virus through phase separation, thereby having very positive significance for the infection regulation of HBV, the theoretical research of pathogenic mechanism and the clinical treatment.

Based on the research results, the invention provides the following technical scheme:

in a first aspect of the invention, there is provided the use of ABCF1 or an agonist thereof in the preparation of an anti-DNA virus formulation.

Preferably, the ABCF1 can adopt a commercial recombinant ABCF1 protein preparation, and the source of the recombinant ABCF1 protein preparation is not limited to engineering bacteria expression or liquid phase synthesis, solid phase synthesis and the like.

Further, the ABCF1 also broadly comprises derivative polypeptides formed by chemical or genetic modification of the above polypeptides; the modifier includes, but is not limited to, streptavidin, biotin, radioisotope, fluorescent agent, enzyme, cytotoxic substance, antitumor agent, etc., and also includes products in which the protein is immobilized by physical adsorption, carrier, resin cross-linking material, etc.

Further, the ABCF1 also includes a fusion polypeptide of the ABCF1 protein and an effector fragment, the effector fragment including a cell-penetrating peptide or a fragment for extending the in vivo half-life of ABCF1, such as serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin, and the like.

Further, the ABCF1 also includes a truncation of ABCF1, which retains a partial sequence in ABCF1 and has substantially similar physiological activity to ABCF1.

Preferably, the agonist of ABCF1 includes, but is not limited to, a compound entity, a polymer, a polypeptide or a nucleic acid substance capable of stimulating an increase in the expression level of ABCF1 in the body of a subject, and further includes related agents, such as plasmids, lentiviruses and the like, for overexpression of ABCF1 in the body of a subject based on genetic engineering.

The mechanism research aiming at inhibiting virus replication by ABCF1 shows that ABCF1 can promote the expression of interferon in organisms and is expected to realize broad-spectrum antiviral effect, and the anti-DNA virus preparation is preferably used for medicines, model medicaments, detection reagents and the like for preventing, improving or treating DNA virus infection related diseases; further, specific examples of the DNA virus include Hepatitis B Virus (HBV), smallpox virus, herpes virus, cytomegalovirus, EB virus, varicella-zoster virus.

In one embodiment of the invention, the DNA virus is HBV virus, and the above ABCF1 or an agonist thereof is used for preparing an anti-HBV viral agent.

In the above embodiment, the ABCF1 is an ABCF1 truncation comprising the N-terminal domain of ABCF1, and the amino acid sequence of the truncation is shown in SEQ ID NO. 1 and the nucleotide sequence is shown in SEQ ID NO. 2.

The invention proves that the N-terminal domain of the ABCF1 and cccDNA generated in the HBV virus replication process have good binding property, and the replication of HBV virus in a body can be effectively reduced through the binding effect, so that related diseases caused by HBV infection, such as hepatitis B, cirrhosis, hepatic fibrosis, liver cancer, upper gastrointestinal hemorrhage, acute renal dysfunction and the like, can be inhibited.

In a second aspect of the invention, there is provided a pharmaceutical composition comprising an active dose of ABCF1 or an agonist thereof.

Preferably, the pharmaceutical composition further comprises a pharmaceutically necessary carrier.

Preferably, the dosage form of the pharmaceutical composition is not particularly limited, and a solid preparation or a liquid preparation is preferable; in the case of solid formulations, the carrier includes, but is not limited to, excipients, lubricants, binders, disintegrants, and the like; in the case of liquid preparations, the carrier includes without limitation solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like, and preservatives, antioxidants, coloring agents, sweetening agents and other preparation additives may be appropriately added as needed.

As preferred examples of the excipient, for example, lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. can be used.

As preferable examples of the lubricant, for example, magnesium stearate, calcium stearate, talc, colloidal silicon dioxide, and the like can be used.

As preferable examples of the binder, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, and the like can be used.

As preferable examples of the disintegrant, for example, starch, carboxymethyl cellulose calcium, croscarmellose sodium, sodium carboxymethyl starch, and the like can be used.

As preferable examples of the solvent, water for injection, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil and the like can be used.

As preferred examples of the solubilizer, for example, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like are used.

Suitable examples of the suspending agent include, for example, surfactants (stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, etc.), hydrophilic polymers (polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethylcellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, etc.) may be used.

As suitable examples of tonicity agents, for example, sodium chloride, glycerin, D-mannitol and the like can be used.

As preferable examples of the buffer, for example, a buffer solution of phosphate, acetate, carbonate, citrate, or the like can be used.

As a preferable example of the soothing agent, for example, benzyl alcohol or the like can be used.

As preferable examples of the preservative, for example, paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like can be used.

As preferable examples of the antioxidant, for example, sulfite, ascorbic acid and the like can be used.

Preferably, the pharmaceutical composition further comprises other antiviral active ingredients or auxiliary antiviral ingredients, and the antiviral active ingredients are preferably anti-DNA virus drugs, such as acyclovir; the auxiliary antiviral components include but are not limited to immunoregulation medicines, antibiotics, antipyretics and medicines for relieving respiratory urgency, such as aminopyrine, antongidine, penicillin, streptomycin, nikkomyl, aminophylline, etc.

In a third aspect of the present invention, there is provided a therapeutic agent for a disease associated with HBV viral infection, wherein the therapeutic agent comprises an active dose of ABCF1, ABCF1 truncation or an agonist of ABCF1.

In the third aspect, the HBV virus infection-associated disease is preferably hepatitis B or liver cancer (HCC).

In addition, the dose of the above-mentioned ABCF1, ABCF1 truncated body or ABCF1 agonist is a technique that can be conventionally confirmed depending on the purpose of administration of the therapeutic drug, the subject, and the mode of administration.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Fig. 1 ND domain of ABCF1 is a key domain for binding cccDNA;

a: the ability of ABCF1 to bind to cccDNA in different models;

b: comparing the binding capacity of different truncations of ABCF1 and cccDNA;

c: GST-Pull down experiment analyzes the binding level of the ABCF1-ND protein and cccDNA;

d: in vitro Biotin-pull down demonstrated the binding of ABCF1-ND protein to cccDNA

FIG. 2 shows that ABCF1 mainly depends on its ND domain for suppressing HBV replication;

a: ABCF1 inhibits HBV replication in the transfection model;

b: ABCF1 inhibits HBV replication in a viral infection model;

c: the effect of different truncations of ABCF1 on HBV replication;

FIG. 3 is an IFN response of ABCF1 to promote HBV infection;

a: the levels of IFN- β, IFN- λ, CXCL10 that ABCF1 promotes HBV infection in the transfection model;

b: antiviral effects of ABCF1 in IFNAR-KO cell line;

FIG. 4 shows that the lysine (K) repeat domain and the glutamine (Q) repeat domain of ABCF1-ND are key regions for phase separation of ABCF1 and suppression of HBV replication;

a: in vitro GFP-ABCF1, GFP-ABCF1-ND and GFP-ABCF 1-delta ND phase separation ability and Cy5-MC-HBV fusion ability analysis;

b and C: comparison of antiviral abilities of ABCF1-ND repeat sequence prediction (B) and ND deletion mutant (C);

d: comparison of the levels of phase separation ability of ND, ND-. DELTA.1 KK, ND-. DELTA.Q and ND-. DELTA.1K/Q;

FIG. 5 shows that ABCF1 inhibits the binding of host RNA polymerase II to cccDNA by phase separation;

a: effect of ABCF1 on binding of RNA polymerase II to cccDNA in different models;

b: the effect of ND, ND-. DELTA.1 KK, ND-. DELTA.Q and ND-. DELTA.1K/Q on the binding of RNA polymerase II to cccDNA in different models;

fig. 6 is a mechanism by which ABCF1, an interacting protein of cccDNA, can participate in the regulation of interferon response.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

1) ND domain of ABCF1 is the key domain for binding cccDNA

The ABCF1 can be combined with cccDNA by using a Huh7/MC-HBV transfection model, an HLCZ01/HBV virus infection model and a Biotin-pull down experimental technology (A). In the Huh7/MC-HBV transfection model, through the interaction of the ABCF1 truncated body and cccDNA, ND domain of ABCF1 is found to be the key structural domain for binding cccDNA (B). The in vitro prokaryotic expression purification of ABCF1-ND protein, GST-pull down experiment proves that ABCF1-ND can be directly combined with cccDNA (C), and in vitro Biotin-pull down experiment further proves that ABCF1-ND can be combined with cccDNA (D), which indicates that ND domain is the key structural domain of ABCF1 combined with cccDNA.

2) ABCF1 inhibits HBV replication mainly depending on its ND domain

By using HepG2/MC-HBV transfection model and HepaRG/HBV virus infection model, the over-expression ABCF1 is found to be capable of obviously inhibiting the levels of HBsAg, HBeAg, pgRNA, HBV DNA and HBc of HBV, and the interference ABCF1 is capable of obviously promoting the replication levels (A and B) of HBV. Through antiviral experiments of ABCF1 truncated body on HBV, ND domain of ABCF1 is found to be a key structural domain (C) for inhibiting HBV replication.

3) ABCF1 promotes HBV infection-mediated IFN response

In HepG2/MC-HBV transfection model, it was found that ABCF1 can significantly promote the levels of IFN-. Beta.IFN-. Lambda.CXCL 10 (A) when HBV exists. Using IFNAR-KO cell line, it was found that when IFN receptor is knocked out, inhibitory effect of ABCF1 on HBV still exists (B), suggesting that ABCF1 can promote IFN response of HBV infection, but its antiviral effect may not be completely dependent on IFN.

4) The lysine (K) repeat domain and the glutamine (Q) repeat domain of ABCF1-ND are key regions for phase separation of ABCF1 and inhibition of HBV replication

In vitro prokaryotic expression of purified GFP-ABCF1-FL, GFP-ABCF1-ND and GFP-ABCF1- Δ ND proteins revealed that GFP-ABCF1 protein could be fused with Cy 5-labeled MC-HBV, phase separation occurred, and ND formed phase separation more strongly than ABCF1 (A). The website is used for predicting the repetitive sequence in the ND fragment, an ND deletion mutant is constructed, and the antiviral effects of ND-delta 1KK and ND-delta Q are obviously weakened (B). The GFP-ND, GFP-ND-delta 1KK, GFP-ND-delta Q and GFP-ND-delta 1K/Q proteins are purified by in vitro prokaryotic expression, and compared with ND, the phase separation level and the antiviral effect of ND-delta 1KK, ND-delta Q and ND-delta 1K/Q are obviously weakened (C and D), which indicates that a lysine (K) repeat domain and a glutamine (Q) repeat domain of ABCF1-ND are key areas for phase separation of ABCF1 and inhibition of HBV replication.

5) ABCF1 inhibits the binding of host RNA polymerase II to cccDNA by phase separation

Using the Huh7/MC-HBV transfection model and the HLCZ01/HBV virus infection model, it was found that ABCF1 can inhibit the binding of host RNA polymerase II to cccDNA and is dependent on ND (A). Also using the above model, it was found that when the lysine (K) repeat domain and glutamine (Q) repeat domain of ND were deleted, i.e., ND- Δ 1KK, ND- Δ Q and ND- Δ 1K/Q, the ability to inhibit the binding of RNA pol II to cccDNA was weakened (B), suggesting that ABCF1 inhibits the binding of host RNA polymerase II to cccDNA by phase separation.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

Use of ABCF1 or an agonist thereof for the preparation of an anti-DNA virus preparation.
2. The use of ABCF1 or an agonist thereof for preparing an anti-DNA virus preparation according to claim 1, wherein the ABCF1 can be prepared from a commercially available recombinant ABCF1 protein preparation, and the source of the preparation is not limited to the expression of engineering bacteria or the manner of liquid phase synthesis or solid phase synthesis.
3. Use of ABCF1 or an agonist thereof as defined in claim 1 for the preparation of an anti-DNA virus agent, wherein the ABCF1 further comprises a polypeptide formed by chemical or genetic modification of said polypeptide; the modifier comprises but is not limited to streptavidin, biotin, radioactive isotope, fluorescent agent, enzyme, cytotoxic substance and antitumor agent, and also comprises a product of the protein which is fixed by adopting physical adsorption, carrier and resin cross-linking material;

or, the ABCF1 further comprises a fusion polypeptide of the ABCF1 protein with an effector fragment comprising a cell-penetrating peptide or a fragment for extending the in vivo half-life of ABCF1, such as serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin;

or, the agonist of ABCF1 is a compound entity, polymer, polypeptide or nucleic acid substance capable of stimulating the increase of the expression level of ABCF1 in the body of the subject, and also includes related agents, such as plasmids and lentiviruses, for over-expressing ABCF1 in the body of the subject based on genetic engineering.
4. Use of ABCF1 or an agonist thereof for the preparation of an anti-DNA virus preparation according to claim 1, wherein the anti-DNA virus preparation is a medicament, model agent or test agent for the prevention, amelioration or treatment of a disease associated with DNA virus infection; the DNA virus is selected from hepatitis B virus, smallpox virus, herpes virus, cytomegalovirus, EB virus or varicella-zoster virus.
5. Use of ABCF1 or an agonist thereof for the preparation of an anti-DNA virus preparation according to claim 4 wherein the DNA virus is an HBV virus and the use is the use of an ABCF1 truncation comprising the N-terminal domain of ABCF1 for the preparation of an anti-HBV viral preparation.
6. Use of ABCF1 or an agonist thereof for the preparation of an anti-DNA virus formulation according to claim 5, wherein said HBV viral infection related disease includes but is not limited to hepatitis b, cirrhosis, liver fibrosis, liver cancer, upper gastrointestinal bleeding or acute renal insufficiency.
7. A pharmaceutical composition comprising an active dose of ABCF1 or an agonist thereof.
8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier;

or, the dosage form of the pharmaceutical composition is a solid preparation or a liquid preparation; in the case of solid formulations, the carrier includes, but is not limited to, excipients, lubricants, binders, disintegrants; in the case of liquid preparations, the carrier includes without limitation solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, preservatives, antioxidants, coloring agents, sweetening agents and other formulation additives may also be appropriately added as required;

or, the pharmaceutical composition also comprises other antiviral active ingredients or auxiliary antiviral ingredients, and the antiviral active ingredients are anti-DNA virus drugs, such as acyclovir; the auxiliary antiviral component comprises but is not limited to an immunoregulation medicament, an antibiotic, an antipyretic or respiratory urgency relieving medicament.
9. A therapeutic agent for a disease associated with HBV viral infection, comprising an active dose of ABCF1, an ABCF1 truncation or an agonist of ABCF1.
10. The therapeutic agent for HBV infection-associated diseases according to claim 9, wherein said HBV infection-associated disease is hepatitis B or liver cancer.