Compounds inhibiting hepatitis b surface antigen and uses thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and discloses a compound for inhibiting hepatitis B surface antigen and application thereof. It is proved through research that the recombinant plasmid S-HiBiT constructed can characterize the expression and secretion of HBs, and can be used for screening the compound for inhibiting HBs, and a cell model for screening anti-HBV drugs targeting HBsAg, i.e. HepG2-PB-S-HiBiT stable expression cell line. It is verified through experiments that the model is particularly suitable for high-throughput screening of compounds. Compound 1 and compound 3 capable of inhibiting HBsAg are found through screening of a compound library by using the model, the drug has the potential to be further developed into a hepatitis B treatment candidate drug, and a new drug is provided for the treatment of hepatitis B.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, specifically to a compound that inhibits hepatitis B surface antigen and its applications. Background Technology

[0002] Hepatitis B virus (HBV) infection is a global epidemic. Uncontrolled chronic HBV infection can develop into life-threatening end-stage chronic liver diseases, such as cirrhosis and hepatocellular carcinoma, causing nearly 100,000 deaths annually. The reason the body cannot produce antibodies to clear HBV may be that after HBV infection, various different viral particles are produced. Among these, subviral particles (SVP), also known as hepatitis B surface antigen (HBsAg or HBs) particles, are secreted in extremely large quantities, exceeding the infectious HBV particles by more than 10,000 times. SVP is considered a decoy for the immune system, depleting B and T cell responses and thus contributing to persistent viral infection. Clinically, the disappearance of HBsAg or the appearance of anti-HBs antibodies signifies durable immune control or functional cure of HBV infection.

[0003] Currently, clinically used hepatitis B treatments such as interferon-alpha (IFN-α, PEG-IFNα) and six nucleoside (acid) analogs (lamivudine, adefovir, entecavir, telbivudine, tenofovir, and tenofovir alafenamide) can completely suppress serum HBV DNA to undetectable levels in the vast majority of patients, thereby improving liver function and reducing the incidence of cirrhosis and liver cancer. However, these drugs cannot target and clear the surface antigen to achieve functional cure, and due to the difficulty in clearing conjugated closed circular DNA (cccDNA, the template for HBV transcription and replication), clinical cure can only be achieved in a small number of patients. Therefore, clearing HBsAg through treatment is considered key to restoring the host's antiviral immune response and achieving functional cure of chronic hepatitis B. Developing antiviral drugs targeting HBsAg to achieve this therapeutic goal is of great significance.

[0004] Several candidate molecules targeting HBsAg have been identified, including natural products, synthetic compounds, siRNA, and NAP. siRNA and NAP have undergone clinical trials, but due to drawbacks such as the short half-life of RNA interference and its susceptibility to mutational escape, and the unclear safety profile of NAP in patients with cirrhosis, they have not yet been approved for marketing. Therefore, the development of other drugs targeting HBsAg remains necessary. Summary of the Invention

[0005] This invention first constructs a cell model for screening anti-HBV drugs targeting HBsAg, and then uses this model to screen compounds, discovering compounds that can inhibit HBsAg. These drugs have the potential to be further developed into candidate drugs for the treatment of hepatitis B. Based on this, this invention provides the following technical solution:

[0006] The first aspect of the present invention provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of an HBV inhibitor or in the preparation of a medicament for treating hepatitis B virus infection or disease.

[0007]

[0008] A second aspect of the invention provides the use of a compound of Formula III or a pharmaceutically acceptable salt thereof in the preparation of an HBV inhibitor or a medicament for treating hepatitis B virus infection or disease.

[0009]

[0010] In the above-described application, the compound represented by Formula I or Formula III, or its pharmaceutically acceptable salt, inhibits hepatitis B surface antigen. The application is the use of the compound represented by Formula I or Formula III, or its pharmaceutically acceptable salt, in the preparation of a drug that inhibits hepatitis B surface antigen.

[0011] In the above-described application, preferably, the drug further includes a pharmaceutically acceptable carrier.

[0012] A third aspect of the invention provides a pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infection or disease, comprising a compound of formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0013] A fourth aspect of the invention provides another pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infection or disease, comprising the above-described compound of formula III or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0014] The above-described pharmaceutical composition contains an active ingredient that exerts its efficacy by inhibiting hepatitis B surface antigen.

[0015] The pharmaceutical composition is available in the following dosage forms: capsules, tablets, pills, granules, oral solutions, microcapsules, or injections.

[0016] The fifth aspect of the present invention provides the use of the above-described pharmaceutical composition in the preparation of HBV inhibitors or in the preparation of medicaments for treating hepatitis B virus infection or disease.

[0017] In the technical solution described, the active ingredient in the pharmaceutical composition inhibits hepatitis B surface antigen.

[0018] The beneficial effects of this invention are:

[0019] Studies have confirmed that the constructed recombinant plasmid S-HiBiT can characterize the expression and secretion of hepatitis B (HBs) and can be used to screen compounds that inhibit HBs. The HepG2-PB-S-HiBiT stable expression cell line, a cell model for screening anti-HBV drugs targeting HBsAg, has been experimentally verified to be particularly suitable for high-throughput screening of compounds. Using this model, a compound library was screened, and compounds that can inhibit HBsAg were discovered. These drugs have the potential to be further developed into candidate drugs for the treatment of hepatitis B, providing new drugs for the treatment of hepatitis B. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of plasmids S-HiBiT(s), S-HiBiT, HiBiT-S, S-HiBiT-d8-22, and S-HiBiT-R78K.

[0021] Figure 2 These are the screening results for constructing HBs characterization cell models.

[0022] Figure 3 These are the results of S-HiBiT characterization experiments on the expression and secretion of HBs.

[0023] Figure 4 The results of the construction and identification experiments of the HepG2-PB-S-HiBiT stably expressing cell line are shown.

[0024] Figure 5 This is a result of the inhibitory effect of compound 1.

[0025] Figure 6 These are the results of the screening experiment for analogues of compound 1.

[0026] Figure 7 This is a schematic diagram of the structure of plasmid PB-S-HiBiT. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the chemical and biological reagents used are conventional reagents in the art unless otherwise specified.

[0029] The main sources of reagents and materials used in the embodiments of this application are:

[0030] 2×PrimeSTARHS Mix: Takara Corporation, Japan;

[0031] Gel recovery kit, genomic DNA extraction kit: QIAGEN, Germany;

[0032] Top 10 E. coli strains; Nano Glo HiBiTLytic Detection System: Promega, USA;

[0033] BsmB I, Tangobuffer, DTT, Puromycin: Thermo Scientific, USA;

[0034] Compound library: This is a mini compound skeleton library (Mini skeleton library | TargetMol) purchased from Shanghai Taoshu Biotechnology Co., Ltd. (China);

[0035] ATP, T7 ligase, T4 ligase buffer, T4 polynucleotide kinase: New England Biolabs, USA;

[0036] The plasmid template pCH9 / 3091 was constructed by Michael Nassal of the University of Freiburg, Germany, and is described in Chinese patent application CN202110351152.6 (publication number CN 113025651 B);

[0037] Southern blot, Northern blot detection kits, reverse transcription kits, and qPCR kits: Roche, Germany;

[0038] HBV surface antigen and E antigen detection kit: Shanghai Kehua Bioengineering Co., Ltd., China;

[0039] HepG2 cells: US Type Culture Collection Center.

[0040] The amplification primer sequences used in the embodiments of this invention are shown in Table 1 below:

[0041] Table 1. Primer sequences used in the embodiments of the present invention

[0042]

[0043]

[0044] Example 1: Construction of a cell model for drug screening targeting HBsAg

[0045] 1. Design concept and working principle

[0046] Screening for drugs targeting HBsAg requires a simple and reliable cell screening system. Currently, commonly used cell systems for HBV research include non-infected cell models, infected models, and recombinant cccDNA transfected cell models. Non-infected models include transient transfection cell models and stable transfection models. Transient transfection models include HepG2 and Huh7 cells transfected with HBV replication plasmids, while stable integration models include HepG2.2.15, HepAD38, HepDE19, and HepBHAe82 cells. Infected models include HepaRG, HepG2-NTCP, and PHH cell models. Theoretically, all these cell models can be used for drug screening targeting surface antigens, but they require ELISA detection, which is relatively cumbersome and time-consuming, making it difficult to meet the requirements of high-throughput screening of compound libraries. Secondly, previous screenings in these cell models often only detected HBs in the culture supernatant, without detecting intracellular HBs, thus failing to provide information on the drug's action mechanism (such as its effect on expression or secretion).

[0047] To address these issues, we designed and constructed a simple and effective HBs characterization model based on the NanoLuc luciferase complementary reporter system (NanoBiT). This model primarily utilizes an expression tag derived from NanoLuc (NLuc) luciferase to characterize HBs expression and secretion by measuring luciferase activity in intracellular and culture supernatants. NLuc is a novel luciferase that offers advantages such as better stability and higher luminescence intensity compared to other luciferases. NanoBiT is a two-component complementary system composed of two subunits: High NanoBiT (HiBiT) and Large NanoBiT (LgBiT). While neither subunit possesses enzymatic activity individually, the complex restores its nanoluciferase activity when HiBiT and LgBiT are bound intracellularly or in vitro. The HiBiT tag, only 11 amino acids long, functions similarly to a bright luciferase, enabling simple and sensitive quantification of tagged proteins within a linear dynamic range of seven orders of magnitude, due to its wide and sensitive dynamic range. Researchers have developed a novel luciferase complementary reporter system (NanoBiT) based on NLuc for protein quantification. In this system, NLuc is divided into two parts: an 11-amino acid polypeptide (HiBiT) and an approximately 13 kDa (LgBiT) polypeptide. Neither LgBiT nor HiBiT possesses luciferase activity individually, but they exhibit high affinity (Kd = 700 pM). When they bind together, luciferase activity is restored. If a target protein is tagged with HiBiT, this tag can be detected by LgBiT, thus reflecting the amount of the target protein. The NanoBiT system offers two significant advantages: first, the small size of HiBiT helps maintain the normal structure and function of the target protein; second, the high luciferase activity of the NLuc recovered through complementation enhances detection sensitivity.

[0048] Our constructed HiBiT-HBs fusion model mainly consists of HBs expressed driven by the SP2 promoter, the luciferase tag HiBiT, and an ampicillin resistance gene. After transfection with this plasmid, HiBiT and HBs are stably expressed, and HBs can be characterized by HiBiT. Due to the simplicity and speed of HiBiT tag detection, this model is particularly suitable for high-throughput screening of compounds.

[0049] 2. Model construction and identification of HBs using HiBiT

[0050] To screen suitable HBsAg characterization methods, we constructed three different plasmids fused with the HiBiT tag: SP2-preS2-S-G4S-HiBiT (S-HiBiT(s)), SP2-preS2-S-3XG4S-HiBiT (S-HiBiT), and SP2-preS2-HiBiT-15XG4S-S (HiBiT-S). All three plasmids expressed the S protein fused with the HiBiT tag under the control of the HBVpreS2 promoter. The HiBiT tags were located at the C-terminus or N-terminus of HBs, and separated by glycine-serine (GS) linkers of different lengths. Figure 1 Then, mutations were introduced into the S-HiBiT plasmid, which showed high expression levels of both HBs protein and the HiBiT tag in all three plasmids, for further identification. The fusion of HBs and the HiBiT tag was then tested to determine whether it affected the formation and secretion of SVP, and the response to HBs inhibitors.

[0051] (1) Construction of plasmid SP2-preS2-S

[0052] Using plasmid PCH9 / 3091 as a template, the SP2-preS2-S fragment was amplified with primer F 2925-GAAT+R HBV 1986. The reaction system consisted of: 10 ng of plasmid PCH9 / 3091, 1 μl each of primer F 2925-GAAT (10 μM) and RHBV 1986 (10 μM), 25 μl of 2X PrimeSTAR HS Mix, and sterile ultrapure water to a final volume of 50 μl. The reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag1.

[0053] Using plasmid PCH9 / 3091 as a template, the vector fragment was amplified using primers FSV40 GG2+R CMV GG. The reaction mixture consisted of 10 ng of plasmid PCH9 / 3091, 1 μl each of primers FSV40 GG2 (10 μM) and R CMV GG (10 μM), 25 μl of 2X PrimeSTARHSMix, and sterile ultrapure water to a final volume of 50 μl. The reaction conditions were: 95℃ pre-denaturation for 5 min; 35 cycles of 94℃ for 20 s, 56℃ for 15 s, and 72℃ for 45 s. The amplified fragment was recovered using a gel extraction kit and named frag2.

[0054] The two fragments frag1 and frag2 obtained above were subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0055] <![CDATA[H2O]]> 2μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag1 3μl (100ng) frag2 1 μl (50 ng) Total volume 10μl

[0056] Reaction conditions: 37℃ for 5 min, 20℃ for 5 min, 25 cycles. Inactivation reaction at 80℃ for 20 min.

[0057] Golden Gate product was transformed into TOP10 competent bacteria, plated, cloned, sequenced and identified, and the correct clone was named plasmid SP2-preS2-S.

[0058] (2) Construction of plasmid S-HiBiT(s)

[0059] Using plasmid SP2-preS2-S as a template, the SP2-preS2-S-HiBiT(s) fragment was amplified with primers FS 822+RS 821. The reaction system consisted of: 10 ng of plasmid SP2-preS2-S, 1 μl each of primers FS 822 (10 μM) and RS 821 (10 μM), 25 μl of 2×PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag3.

[0060] The obtained fragment frag3 was subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0061] <![CDATA[H2O]]> 4μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag3 2μl (100ng) Total volume 10μl

[0062] Reaction conditions: 37℃ for 5 min, 20℃ for 5 min, 25 cycles. Inactivation reaction at 80℃ for 20 min.

[0063] Golden Gate product was transformed into TOP10 competent bacteria, plated, cloned, sequenced and identified, and the correct clone was named plasmid S-HiBiT(s).

[0064] (3) Construction of plasmids S-HiBiT and HiBiT-S

[0065] Using plasmid S-HiBiT(s) as a template, the SP2-preS2-S-HiBiT fragment was amplified with primers F HiBiT-G4S+RFHiBiT-G4S. The reaction system consisted of: 10 ng of plasmid SP2-preS2-S-HiBiT, 1 μl each of primers F HiBiT-G4S (10 μM) and RFHiBiT-G4S (10 μM), 25 μl of 2X PrimeSTAR HS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag4.

[0066] Using plasmid S-HiBiT(s) as a template, the SP2-preS2-S fragment was amplified with primers Fs+Rs. The reaction system consisted of 10 ng of plasmid SP2-preS2-S, 1 μl each of primers Fs (10 μM) and Rs (10 μM), 25 μl of 2X PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. The reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag5. Using plasmid HiBiT-DDB1 as a template, the HiBiT fragment was amplified with primers F HiBiT-G4S+RHiBiT-G4S. The reaction system consisted of: 10 ng of plasmid HiBiT-DDB1, 1 μl each of primers F HiBiT-G4S (10 μM) and RHiBiT-G4S (10 μM), 25 μl of 2X PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag6.

[0067] The obtained fragment frag4 was subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0068] <![CDATA[H2O]]> 4μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag4 2μl (100ng) Total volume 10μl

[0069] The two fragments frag5 and frag6 obtained above were subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0070] <![CDATA[H2O]]> 2μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag5 2μl (100ng) frag6 2μl (100ng) Total volume 10μl

[0071] Reaction conditions: 37℃ for 5 min, 20℃ for 5 min, 25 cycles. Inactivation reaction at 80℃ for 20 min.

[0072] Golden Gate product was transformed into TOP10 competent bacteria, plated, cloned, sequenced and identified, and the correct clones were named plasmids S-HiBiT and HiBiT-S.

[0073] (4) Screening for expression of plasmids S-HiBiT(s), S-HiBiT, and HiBiT-S

[0074] The HiBiT tag, upon binding to LgBiT, exhibits luciferase activity, catalyzing the luminescence of the corresponding substrate, thus providing a convenient method for the detection of target proteins. To test whether the HiBiT tag can be used to characterize hepatitis B (HBs), we first constructed plasmids expressing S-HiBiT(s), S-HiBiT, and HiBiT-S, respectively. Figure 1 Of the three plasmids, HiBiT is located at the C-terminus or N-terminus of HBs, separated by glycine-serine (GS) linkers of different lengths. After transfecting HepG2 cells with the three plasmids, HBsAg and HiBiT tags were detected in intracellular and culture supernatants after 48 hours. ELISA and Western blot (anti-S antibody) results showed that HBs protein was expressed in cells transfected with all three plasmids. Cells transfected with S-HiBiT(s) and S-HiBiT showed high levels of S protein expression in both intracellular and culture supernatants, while HiBiT-S showed low intracellular expression and was virtually undetectable in the culture supernatant. Figure 2 A). Nluc activity and Western blot results (using lgBiT to detect HiBiT-S fusion protein on the membrane) showed that HiBiT tags were expressed in cells transfected with all three plasmids. Cells transfected with S-HiBiT showed high levels of HiBiT tag expression both intracellularly and in the culture supernatant; cells transfected with S-HiBiT(s) showed detectable HiBiT tags both intracellularly and in the culture supernatant, but at lower levels than those transfected with S-HiBiT; while HiBiT-S showed high intracellular expression levels, it was virtually undetectable in the culture supernatant. Figure 2 B). Therefore, we will further identify plasmid S-HiBiT, which has high expression levels of both HBs protein and the HiBiT tag.

[0075] (5) Construction of plasmids S-HiBiT-d8-22 and S-HiBiT-R78K

[0076] Using plasmid S-HiBiT as a template, the SP2-preS2-S fragment mutant was amplified with primers FS R78K+RS R78K. The reaction system consisted of: 10 ng of plasmid S-HiBiT, 1 μl each of primers FS R78K (10 μM) and FS R78K (10 μM), 25 μl of 2X PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag7.

[0077] Using plasmid S-HiBiT as a template, the HiBiT fragment was amplified using primers FS(d8-22) + RS(d8-22). The reaction system consisted of: 10 ng of plasmid S-HiBiT, 1 μl each of primers FS(d8-22) (10 μM) and RS(d8-22) (10 μM), 25 μl of 2X PrimeSTARHSMix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag8.

[0078] The obtained fragment frag7 was subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0079] <![CDATA[H2O]]> 5μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag7 1 μl (150 ng) Total volume 10μl

[0080] The obtained fragment frag8 was subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0081] <![CDATA[H2O]]> 4μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag8 2μl (100ng) Total volume 10μl

[0082] Reaction conditions: 37℃ for 5 min, 20℃ for 5 min, 25 cycles. Inactivation reaction at 80℃ for 20 min.

[0083] Golden Gate product was transformed into TOP10 competent bacteria, plated, cloned, sequenced and identified, and the correct cloning names were plasmids S-HiBiT-d8-22 and S-HiBiT-R78K.

[0084] (6) Identification of plasmid S-HiBiT

[0085] We introduced two mutations into pS-HiBiT to observe their effects on its secretory behavior. The first mutation was the deletion of amino acids 8-22 of HBs (in the previously constructed plasmid S-HiBiT-d8-22). This deletion prevents HBs from being transported to the endoplasmic reticulum and thus prevents secretion. The second mutation was the mutation of arginine (R) at position 78 of HBs to lysine (K) (in the previously constructed plasmid S-HiBiT-R78K). The amino acid sequence of wild-type HBs is shown in SEQ ID NO. 23.

[0086] The amino acid sequence (SEQ ID NO.23) of wild-type HBs is as follows:

[0087] MENITSGFLGPLLVLQAGXFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGS STTSTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI.

[0088] Since HBs binds to SEC24A via the R78 / R79 dipeptide signaling pathway and then leaves the endoplasmic reticulum via the capsid protein complex II (COPII) vesicle transport mechanism, mutants containing R78K cannot leave the endoplasmic reticulum to complete secretion. HepG2 cells were transfected with pS-HiBiT-d8-22 and pS-HiBiT-R78K, respectively. HBsAg and HiBiT levels were detected intracellularly and in the culture supernatant after 48 hours. ELISA and Western blot (anti-S antibody) results showed that intracellular HBs expression levels were unaffected after transfection with both mutants, but HBs could not be detected in the culture supernatant. Figure 3 A). Nluc activity and Western blot (LgBiT incubation) assays showed that intracellular HiBiT tag expression levels were unaffected after mutation, while the supernatant was significantly reduced to near-background levels. Figure 3 B). Literature reports that DNAJB12 and SEC24A are involved in the secretion of HBsAg. We transfected pS-HiBiT into HepG2 cells with knocked-down SEC24A and DNAJB12, and detected HBsAg and HiBiT tags in the culture medium using ELISA and a HiBiT assay kit. The results showed reduced HBsAg secretion in the culture supernatant, along with decreased Nluc activity. Figure 3C). In summary, pS-HiBiT can normally express the HBs-HiBiT fusion protein, and the detection of HiBiT can indicate HBs.

[0089] To test whether the fusion of HBs with the HiBiT tag affects the formation and secretion of SVP, HepG2 cells were transfected with pHBV1.3, pS-HiBiT-d8-22, and pS-HiBiT, respectively. SVP in the culture supernatant and intracellular samples were then detected using a particulate gel electrophoresis method. The results showed that SVP could be detected using both lgBiT and anti-S-labeled monoclonal antibodies. Figure 3 D).

[0090] BFA is a fungal compound that has been reported to inhibit HBs secretion. We tested the effect of BFA on S-HiBiT secretion. HepG2 cells transfected with pS-HiBiT were treated with BFA at different time points (0h, 6h, 12h, 24h) and at different concentrations (0nM, 10nM, 100nM, 1uM, 40uM) for 24h. HBsAg and HiBiT tags in the culture supernatant were then measured. The results showed that the levels of HBs and HiBiT in the culture supernatant were inhibited by BFA, and this inhibitory effect was time-dependent. Figure 3 E) and dose-dependent ( Figure 3 F). Therefore, S-HiBiT can reflect the effect of inhibitors on HBs.

[0091] In summary, S-HiBiT can characterize the expression and secretion of hepatitis B (HBs) and can be used to screen for compounds that inhibit HBs.

[0092] 3. Construction and identification of HepG2-PB-S-HiBiT stable expression cell lines

[0093] To rapidly screen a library of compounds targeting HBsAg, a transposon plasmid PB-SPII-Pres2-S-HiBiT-pEF1α-mCherry-2APuro (PB-S-HiBiT for short) was constructed based on the validated pS-HiBiT. After co-transfection with the piggybac transposase expression plasmid PB-PA200-1, single cell clones were selected by puromycin selection and expanded to construct a stable HepG2-PB-S-HiBiT expression cell line. The expanded cells were then identified.

[0094] (1) Construction of plasmid PB-S-HiBiT

[0095] Using plasmid PB-pCMV-NTCP-pEF1α-mCherry-2APuro as a template, a DNA fragment was amplified using primers R cmv1442 ATTC + FPB513B5600. The reaction mixture consisted of: 10 ng plasmid PB-pCMV-NTCP-pEF1α-mCherry-2APuro, 1 μl each of primers R cmv1442ATTC (10 μM) and FPB513B5600 (10 μM), 25 μl of 2×PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 56℃ for 15 s, 72℃ for 1 min, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag9.

[0096] Using plasmid PB-pCMV-NTCP-pEF1α-mCherry-2APuro as a template, a DNA fragment was amplified using primers RPB513B 5603+FpEF-1α1785TAAC. The reaction mixture consisted of: 10 ng of plasmid PB-pCMV-NTCP-pEF1α-mCherry-2APuro, 1 μl each of primers RPB513B 5603 (10 μM) and FpEF-1α1785TAAC (10 μM), 25 μl of 2×PrimeSTAR HS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 56℃ for 15 s, 72℃ for 1 min, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag10.

[0097] Using plasmid S-HiBiT as a template, the SP2-preS2-S fragment was amplified with primers F 2925-GAAT+RHBV 1986. The reaction system consisted of: plasmid PCH9 / 309110 ng, 1 μl each of primers F 2925-GAT (10 μM) and RHBV 1986 (10 μM), 25 μl of 2×PrimeSTARHS Mix, and sterile ultrapure water to a final volume of 50 μl. Reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min 30 s, for 35 cycles. The amplified fragment was recovered using a gel extraction kit and named frag11.

[0098] The three fragments frag9, frag10, and frag11 obtained above were subjected to a Golden Gate linkage reaction. The reaction system was as follows:

[0099] <![CDATA[H2O]]> 1μl BsmBI enzyme 0.75μl Tangobuffer 1μl DTT 1μl T7ligase 0.25μl ATP 1μl frag9 1 μl (50 ng) frag10 2μl (30ng) frag11 2μl (30ng) Total volume 10μl

[0100] Reaction conditions: 37℃ for 5 min, 20℃ for 5 min, 25 cycles. Inactivation reaction at 80℃ for 20 min.

[0101] The Golden Gate product was transformed into TOP10 competent bacteria, plated, screened for initial cloning, and sequenced for identification. The correct clone was named plasmid PB-S-HiBiT, and its nucleotide sequence is shown in SEQ ID NO.24. The plasmid structure diagram is shown below. Figure 7 As shown.

[0102] (2) Construction and identification of HepG2-PB-S-HiBiT stably expressing cells

[0103] The successfully constructed pPB-S-HiBiT and piggybac transposase expression plasmid PB-PA200-1 were co-transfected into HepG2 cells at a ratio of PB-S-HiBiT:PB-200PA-1 = 5:1. The medium was changed after 12 hours, and after 48 hours, puromycin was added to a final concentration of 2 μg / ml to screen for cells stably expressing HBs-HiBiT. After two weeks of continuous screening, single cells were picked and expanded in culture. Figure 4 A).

[0104] To test whether the S-HiBiT gene fragment was inserted into HepG2 cells, amplified HepG2-PB-S-HiBiT cells and negative controls (HepG2 cells) were collected. Cellular DNA was extracted, and PCR amplification was performed using primers F 2925-GAAT+RHBV 1986. The products were then subjected to gel electrophoresis. The results showed that HepG2-PB-S-HiBiT amplified a band of approximately 2kb, while the negative control (HepG2 cells) showed no band. Figure 4 B). Culture supernatant was collected and HBsAg and HiBiT tags were detected. ELISA and Nluc activity results showed that the constructed PB-S-HiBiT cell line expressed significantly higher levels of HBsAg and HiBiT than the negative control (HepG2 cells). Western blot (LgBiT incubation) results showed that the PB-S-HiBiT cell line expressed HiBiT, while the negative control (HepG2 cells) did not express HiBiT. Figure 4 C). Fluorescence microscopy was used to observe the expression of fluorescent proteins in HepG2-PB-S-HiBiT cells. The results showed red fluorescent cell clusters (C). Figure 4 D). These results indicate that the S-HiBiT gene fragment is inserted into the DNA of HepG2 cells and can express HBsAg and HiBiT normally.

[0105] To test whether the insertion of the S-HiBiT gene fragment affects the formation and secretion of SVP, culture supernatants from HepG2 cells and HepG2-PB-S-HiBiT cells were collected, and SVP in the culture supernatant was detected using a particulate gel electrophoresis method. The results showed that SVP could be detected after incubation with lgBiT. Figure 4 E). In summary, the insertion of the S-HiBiT gene fragment can support the formation and secretion of SVP.

[0106] RG7834 is a dihydroquinolone (DHQ) compound, a first-class, highly selective, orally bioavailable hepatitis B virus inhibitor with a mechanism of action that reduces viral antigen and viral DNA. AD80 is a compound discovered in the inventors' laboratory that inhibits the transcriptional activity of the HBV core promoter, thereby reducing the amount of HBV mRNA (see Chinese Patent ZL202210849550.5). We tested the effects of AD80, RG7834, and BFA on HepG2-PB-S-HiBiT cells. HepG2-PB-S-HiBiT cells were treated with AD80, RG7834, and BFA. Simultaneously, cells were treated with different concentrations of BFA (0 nM, 10 nM, 100 nM, 1 μM, 40 μM) for 24 h, and the levels of HBsAg and HiBiT tags in the culture supernatant were measured. The results showed that the levels of HBsAg and HiBiT in the culture supernatant were inhibited by AD80, RG7834, and BFA. Figure 4 F), and this inhibitory effect of BFA is dose-dependent. Figure 4 G). Therefore, HepG2-PB-S-HiBiT cells can reflect the effect of inhibitors on HBs.

[0107] These results indicate that the HepG2-PB-S-HiBiT stably expressing cell line was successfully constructed and can be used to screen drugs targeting HBsAg.

[0108] Example 2: Screening of compounds targeting HBV transcription

[0109] 1. Initial screening of compounds

[0110] After obtaining stable passaged HepG2-PB-S-HiBiT cells, they were used for compound screening. The screening procedure was as follows: HepG2-PB-S-HiBiT cells were resuscitated and cultured in 10cm culture dishes using DMEM medium containing 10% fetal bovine serum. When the cells were in good growth condition, they were digested and seeded into 96-well plates. The day after cell seeding, the test compounds were added to the 96-well plates at a final concentration of 10μM, with two replicates for each compound. Approximately 72 hours after drug addition, the supernatant was collected and analyzed using a HiBiT assay reagent. We first screened 5088 small molecule compounds and found 591 compounds that reduced HiBiT activity by 2-fold or more. Then, we repeated the experiment on these 591 compounds and found that 126 compounds again reduced HiBiT activity by 2-fold or more. Then, 126 compounds were further screened. The screening procedure was as follows: HepG2.2.15 cells were revived and cultured in DMEM medium containing 10% fetal bovine serum in 10 cm culture dishes. When the cells were in good growth condition, they were digested and seeded into 48-well plates. The day after cell seeding, the test compounds were added to the 48-well plates at a final concentration of 10 μM, with two replicates for each compound. Approximately 72 hours after drug addition, the supernatant was collected and tested with an HBsAg detection reagent. Forty compounds were found to reduce HBsAg by 2-fold or more. These 40 compounds were then repeated, and eight compounds again reduced HBsAg by 2-fold or more. To further evaluate whether the eight candidate compounds inhibited HBsAg in a dose-dependent manner within a specified concentration range, the level of HBsAg in the supernatant was detected by ELISA. The results showed that among the 40 candidate compounds, compound 1 (1-{4-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]piperazin-1-yl}-2-(3,4-dimethoxyphenyl)ethyl-1-one) exhibited a dose-dependent inhibitory effect on secretory HBsAg. Figure 5 In summary, after three rounds of screening, compound 1 was found to inhibit HBsAg in a concentration-gradient manner. Compound 1 (CAS No.: 1189423-18-4, molecular formula: C) 22 H 23 The chemical structural formula of ClN4O4 is shown in Formula I:

[0111]

[0112] 2. Screening of Compound 1 analogues

[0113] Subsequently, we purchased analogues of compound 1 from Ceramics Company and investigated six structural analogues of compound 1: compound 2 (1-{4-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]piperazin-1-yl}-2-(3,4-dimethoxyphenyl)ethyl-1-one), compound 3 (1-{4-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]piperazin-1-yl}-3-(3,4-dimethoxyphenyl)prop-2-en-1-one), and compound 4 (1-{4-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]piperazine). The inhibitory effects of compounds 1-{4-[3-(4-chlorophenyl)-1,2,4-oxadiazol-5-yl]piperazin-1-yl}-2-(4-methylphenoxy)ethane-1-one, 6-[N-[2-(4-chlorophenyl)ethyl]-3-(3,4-dimethoxyphenyl)-1,2,4-oxadiazol-5-amine] and 7-(2-(4-methoxyphenyl)-1-(4-(3-phenyl-1,2,4-oxadiazol-5-yl)piperazin-1-yl)ethane-1-one) on HBsAg.

[0114] The chemical structural formulas of compounds 2, 3, 4, 5, 6, and 7 are shown in Formulas II, III, IV, V, VI, and VII below:

[0115]

[0116]

[0117] The investigation process was as follows: HepG2.2.15 cells were resuscitated and cultured in 10cm culture dishes using DMEM medium containing 10% fetal bovine serum. When the cells were in good growth condition, they were digested and seeded into 48-well plates. The day after seeding, different test compounds, a positive control (RG7834 10 nM), and a negative control (DMSO) were added to the 48-well plates to a final concentration of 10 μM, with two replicates for each compound. Approximately 72 hours after drug addition, the supernatant was collected and tested using an HBsAg detection reagent. The results showed that compounds 2, 3, 4, and 5 in the analogues still exhibited good inhibitory activity against HBsAg. Figure 6 A). To further evaluate the performance of these five compounds, ELISA was used to detect their dose-dependent inhibition of HBsAg, and CCK8 assay was used to detect their cytotoxicity. The results showed that compound 3 was superior to the other four compounds (A). Figure 6 B and 6C). Finally, we further investigated compound 3, showing a CC50 > 10 μM and IC50 = 2.787 μM, demonstrating a clear distinction between the HBsAg inhibitory effect and cytotoxic properties. Figure 6 D and 6E). To verify the inhibitory effect of compound 3, a Dotblot assay was used to detect the level of HBsAg in the supernatant of HepG2.2.15 cells. DMSO was used as a negative control, 25 nM entecavir (ETV) and 10 nM RG7834 were used as positive controls, and HepG2.2.15 cells were treated with 2 μM, 4 μM and 8 μM of compound 3 for 3 days. The results showed that the level of HBsAg in the cell supernatant decreased in a dose-dependent manner after treatment with compound 3 (D and 6E). Figure 6 F).

[0118] In summary, compound 3 (CAS No.: 932985-37-0, molecular formula: C) 23 H 23 ClN4O4 has a good effect on inhibiting HBsAg and has good application prospects.

Claims

1. The use of the compound represented by Formula I or its pharmaceutically acceptable salt in the preparation of HBV inhibitors or in the preparation of medicaments for treating hepatitis B virus infection or disease.

2. The use of the compound represented by Formula III or its pharmaceutically acceptable salt in the preparation of HBV inhibitors or medicaments for treating hepatitis B virus infection or disease.

3. The application according to claim 1 or 2, characterized in that: The compound of Formula I or Formula III, or a pharmaceutically acceptable salt thereof, inhibits hepatitis B surface antigen, wherein the application is the use of the compound of Formula I or Formula III, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting hepatitis B surface antigen.

4. The application according to claim 1 or 2, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

5. A pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infection or disease, characterized in that: It contains the compound of formula I in claim 1 as an active ingredient or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infection or disease, characterized in that: It contains the compound of formula III of claim 2 as an active ingredient or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that: The active ingredient exerts its effect by inhibiting hepatitis B surface antigen.

8. The pharmaceutical composition according to claim 5 or 6, characterized in that: The pharmaceutical composition is available in the following dosage forms: capsules, tablets, pills, granules, oral solutions, microcapsules, or injections.

9. The use of the pharmaceutical composition of claim 5 or 6 in the preparation of HBV inhibitors or in the preparation of medicaments for treating hepatitis B virus infection or disease.

10. The application according to claim 9, characterized in that: The active ingredient in the pharmaceutical composition inhibits hepatitis B surface antigen.

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