Micro-chromosome state characterization cell model for drug screening and new application of compound in resisting hepatitis B virus

Abstract

The invention discloses a mini-chromosome state characterization cell model for drug screening. The mini-chromosome state characterization cell model is a transfected cell obtained by co-transfecting a hepatocyte line with prcccDNA-HiBiT-HBx and a plasmid Cre; the invention also discloses an application of the compound C20H14F3N3S or the pharmaceutically acceptable salt thereof in preparation of an HBV (hepatitis B virus) inhibitor or a medicine for treating hepatitis B virus infectious diseases or symptoms. According to the invention, a novel anti-hepatitis B virus small molecule compound is screened by utilizing a constructed cell model, and experiments prove that the novel anti-hepatitis B virus small molecule compound can obviously reduce the expression levels of HBV total RNAs, HBeAg, HBV pgRNA and HBV nucleoprotein.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a minichromosome state characterization cell model for drug screening and a novel application of a compound in resisting hepatitis B virus. Background Art

[0002] Hepatitis B virus (HBV) infection is a major global public health issue, closely related to chronic hepatitis B, cirrhosis, hepatocellular carcinoma and other liver diseases. It is highly contagious, has a high incidence rate and is widely prevalent, posing a serious threat to human health. However, no treatment strategy has been found to completely cure HBV infection.

[0003] HBV is a DNA enveloped virus belonging to the family Hepadnaviridae. HBV virus particles are composed of nucleocapsid and envelope proteins. The HBV genome is a 3.2kb relaxed circular DNA (rcDNA), of which the longer strand is designated as the negative strand (-) and the shorter strand is the positive strand (+). The negative strand is the coding strand, which contains four overlapping open reading frames (ORFs) (preC / C, P, preS / S and X), four promoters (core promoter, PreS1 promoter, PreS2 / S promoter and X promoter) and two enhancers (EnhⅠ and EnhⅡ), which can transcribe four types of RNA (3.5kbPreC / C mRNA, 2.4kbPreS1mRNA, 2.1kbPreS2 / S mRNA and 0.7kbX mRNA). PreC / CORF is responsible for encoding HBeAg and HBcAg; PORF encodes HBV DNA polymerase; PreS / S ORF encodes large (L), medium (M) and small (S) envelope proteins; XORF encodes X protein (HBx).

[0004] Covalently closed circular DNA (cccDNA) is the source of HBV replication and the cause of chronic infection. After cccDNA is formed, it will exist in the form of a free chromosome-like viral minichromosome and be modified by host histones (such as H3 and H4 histones) and non-histones (such as viral HBx, core protein and host epigenetic related proteins). HBV cccDNA forms a minichromosome (a chromosome-like structure formed by the combination of some viral DNA and host histones) in the nucleus of infected liver cells by winding around nucleosomes to simulate the chromosome existence of human genomic DNA. This form allows HBV cccDNA to exist stably in the host cell nucleus and effectively synthesize viral mRNA using the gene transcription regulatory mechanism in the cell nucleus.

[0005] At present, the drugs used clinically for chronic hepatitis B are mainly pegylated interferon α (Peg-IFNα) and nucleoside analogs (NUCs). Since 1998, six nucleoside (acid) analogs have been approved for use in clinical practice, including lamivudine, entecavir, telbivudine, adefovir dipivoxil and tenofovir. However, the huge challenges faced cannot be ignored: interferon plays an important role in the long-term and sustainable inhibition of cccDNA transcription, but its response rate is poor; nucleoside (acid) analogs can inhibit HBV replication by targeting viral RNA-dependent DNA polymerase, but have no direct effect on existing cccDNA minichromosomes, and viral recurrence is common after termination of nucleoside (acid) analogs, even in patients who have reached the end point of treatment. Summary of the invention

[0006] The purpose of the present invention is to address the above problems and provide a new application of a minichromosome state characterization cell model for drug screening and a compound in anti-hepatitis B virus.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a recombinant plasmid prcccDNA-HiBiT-HBx, whose backbone vector is plasmid prcccDNA, and the recombinant plasmid prcccDNA-HiBiT-HBx is obtained by replacing the first 33bp of the N-terminal HBx sequence of the prcccDNA plasmid with a HiBiT tag sequence.

[0009] The recombinant plasmid prcccDNA-HiBiT-HBx is characterized by:

[0010] The first 33 bp sequence of the N-terminal of the HBx sequence is (SEQ ID NO.11):

[0011] 5'-TGTTGTCCTATCCCGCAAATATACATCGTTTCC-3';

[0012] The HiBiT tag sequence is (SEQ ID NO.12):

[0013] 5'-ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATCAGC-3';

[0014] The amino acid sequence of HiBiT protein is VSGWRLFKKIS (SEQ ID NO. 13).

[0015] The construction method of the above-mentioned recombinant plasmid prcccDNA-HiBiT-HBx comprises the following steps: using the prcccDNA plasmid as a template, performing PCR amplification on HiBiT-SF / HiBiT-SR using primers as shown in SEQ ID NO.1 and 2, digesting the amplified product with Dpn I, and efficiently recombining the digested product using a recombinase to realize in vitro circularization of linear DNA, transforming the recombinant product into competent bacteria, picking a single clone, and sequencing the correct clone as the recombinant plasmid prcccDNA-HiBiT-HBx; preferably, the recombinase is Exnase II.

[0016] The second aspect of the present invention provides a minichromosome state characterization cell model for drug screening, wherein the prcccDNA-HiBiT-HBx and plasmid Cre are co-transfected into a liver cell line to obtain transfected cells.

[0017] The third aspect of the present invention provides the use of the above-mentioned minichromosome state characterization cell model in screening anti-hepatitis B virus drugs.

[0018] In the above application, the above prcccDNA-HiBiT-HBx and Cre plasmid are co-transfected into a liver cell line, the transfected cells are digested, and then treated with the selected compound, and the HiBiT activity level is detected after the treatment; preferably, HiBiT detection kit was used to detect the activity level of HiBiT.

[0019] The fourth aspect of the present invention provides the use of a compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of an HBV inhibitor or a drug for treating a hepatitis B virus infectious disease or condition:

[0020]

[0021] The compound represented by formula I or a pharmaceutically acceptable salt thereof inhibits the transcription of hepatitis B virus;

[0022] Preferably, the compound represented by formula I or a pharmaceutically acceptable salt thereof reduces the expression levels of HBV total RNAs, HBeAg, HBV pgRNA and HBV nucleoprotein.

[0023] The fifth aspect of the present invention provides a pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infectious diseases or conditions, comprising as an active ingredient a compound represented by the above formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0024] The product dosage form of the pharmaceutical composition is capsule, tablet, pill, paste, granule, oral solution preparation, microcapsule preparation or injection.

[0025] The beneficial effects of the present invention are:

[0026] The present invention discloses a new model for characterizing the state of minichromosomes based on HiBiT tag and recombinant cccDNA. HBx in the recombinant cccDNA plasmid is labeled with HiBiT tag, and the state of minichromosomes is directly and indirectly characterized through Cre-loxP-chimeric intron system. The activity level of HiBiT can characterize the transcriptional activity of HBV to a certain extent, and can sensitively characterize the expression level of HBx protein. In cultured liver cancer cells, co-transfection of prcccDNA and Cre leads to accumulation of nuclear rcccDNA. Studies have shown that this rcccDNA is supercoiled, heat-resistant, and epigenetically organized into minichromosomes similar to the natural cccDNA of HBV. The cell model of the present invention has the advantages of direct characterization of cccDNA, simple and rapid detection method, and high sensitivity. The small molecule compounds screened out by the model have been verified by the cell infection model to confirm their inhibitory effect on the transcription level of HBV.

[0027] The present invention screened a new small molecule compound against hepatitis B virus using the above cell model. Experimental verification showed that the compound can significantly reduce the expression levels of HBV total RNAs, HBeAg, HBV pgRNA and HBV nucleoprotein, and its effect is better than other compounds screened in the early stage of this laboratory. This provides a new compound for the treatment of hepatitis B and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The vector map of plasmid prcccDNA-HiBiT-HBx (A) and the structural map of recombinant sequence rcccDNA-HiBiT-HBx (B).

[0029] Figure 2 Schematic diagram of the construction of the recombinant plasmid prcccDNA-HiBiT-HBx and its verification in cells: A: Schematic diagram of the construction of the prcccDNA-HiBiT-HBx plasmid and sequencing peak diagram; B: prcccDNA-HiBiT-HBx was transformed into two cell lines, HepG2 and Huh7, and 72 hours later, the cells were HiBiT cleavage detection system was used to detect HiBiT activity level; C: prcccDNA-HiBiT-HBx and Cre were co-transfected into HepG2 and Huh7 cell lines at a ratio of 1:1, and the rcccDNA content was detected by southern blotting after 72 hours; D: prcccDNA-HiBiT-HBx and Cre were co-transfected into Huh7 cell line at a ratio of 1:1, and the immunoprecipitated rcccDNA was detected by real-time fluorescence quantitative polymerase chain reaction of P3 and P4 after 72 hours.

[0030] Figure 3 Schematic diagram of the screening process for anti-HBV drugs using this model and the screening results: A: Flow chart of small molecule compound screening; B: HiBiT cleavage detection system detects the effects of different small molecule compounds on HiBiT activity level; C: RT-qPCR detects the effects of different small molecule compounds on HBV pgRNA expression level.

[0031] Figure 4 To verify that the screened small molecule compounds play an anti-HBV effect in an in vitro infection model: A: structural formula, molecular formula, and molecular weight of compound 10; B: experimental process of HBV infection in HepG2-NTCP cells; C: ELISA analysis of the effect of compound 10 on HBeAg secretion; D: RT-qPCR detection of the effect of compound 10 on the expression level of HBV pgRNA in HepG2-NTCP; E: RT-qPCR detection of the effect of compound 10 on the expression level of total HBV RNAs in HepG2-NTCP; F: Northern blot detection of the effect of compound 10 on the expression level of total HBV RNAs in HepG2-NTCP; G: Western blot detection of the effect of compound 10 on the expression level of HBc protein in HepG2-NTCP.

[0032] Figure 5 The IC50 and CC50 results of compound 10 and control compounds were tested in different hepatocytes. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0035] NLuc is a new type of luciferase, which has the advantages of better stability and higher luminescence intensity compared with other luciferases. NanoBiT is a two-component structural complementation system composed of two subunits, HighNanoBiT (HiBiT) and Large NanoBiT (LgBiT). Based on NLuc, researchers have developed a new luciferase complementation reporter system (NanoBiT) that can be used for protein quantitative detection. In this system, NLuc is divided into two parts, one of which is a polypeptide with a length of 11 amino acids (HiBiT) and the other has a molecular weight of about 13KD (LgBiT). LgBiT and HiBiT do not have luciferase activity alone, but they have high affinity (Kd = 700pM). When the two are combined together, luciferase activity will be restored. If a target protein is tagged with a HiBiT, the tag will be detected by LgBiT, thereby reflecting the amount of the target protein. The NanoBiT system has two significant advantages: first, HiBiT is tiny in size, which helps maintain the normal structure and function of the target protein; second, the NLuc luciferase activity restored by complementation is very high, which helps improve detection sensitivity.

[0036] The main reagents and materials used in the examples of this application come from:

[0037] Plasmid template prcccDNA: constructed and donated by Shanghai Pasteur Institute, Chinese Academy of Sciences, and recorded in Chinese patent CN201310280198.9 (Announcement No. CN104278055B);

[0038] II Fast Mutagenesis Kit V2: C214 (NoviZan, China), which contains Phanta Max Super-Fidelity DNA Polymerase, Exnase II, and DpnI enzymes;

[0039] HBeAg antigen test kit: Shanghai Kehua Bioengineering Co., Ltd., China;

[0040] 5× SDS-PAGE buffer: Yisheng, China;

[0041] Lipofectamine TM 3000 transfection reagent: L3000015, Thermo Fisher, USA;

[0042] Trizol reagent: Thermo Fisher, USA;

[0043] HiBiT detection kit: N3030, Promega, USA;

[0044] Southern blot, Northern blot detection kit, qPCR kit: Roche, Germany;

[0045] Gel recovery kit: Novezan, China;

[0046] Compound library: purchased from Shanghai Taoshu Biotechnology Co., Ltd. (China);

[0047] IgG antibody: Cell Signaling Technology (#2729), United States;

[0048] H3 antibody: Active Motif (39763), United States;

[0049] H3Ac antibody: Millipore (06-599), United States;

[0050] H3K9Ac antibody: Abclonal (A7255), China;

[0051] H3K27Ac antibody: Abclonal (A7253), China;

[0052] β-actin antibody: Abclonal (AC026), China;

[0053] HBc antibody: kindly donated by Professor Cai Xuefei of Chongqing Medical University.

[0054] Plasmid pCMV-Cre: An expression plasmid of Cre Recombinase containing the CMV promoter, kindly donated by Professor Ren Fang of Chongqing Medical University.

[0055] The PCR primer sequences used in the embodiments of the present invention are as follows:

[0056]

[0057]

[0058] Example 1

[0059] 1. Construction of recombinant plasmid prcccDNA-HiBiT-HBx.

[0060] HBx is the smallest open reading frame encoding on the HBV genome, consisting of 154 amino acid residues. HBx is an important non-structural protein with transactivation effect.

[0061] First, we replaced the first 33 bp of the N-terminal HBx sequence of the plasmid template prcccDNA (SEQ ID NO.11: 5'-TGTTGTCCTATCCCGCAAATATACATCGTTTCC-3') with HiBiT tag (SEQ ID NO.12: 5'-ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATCAGC-3') to design the prcccDNA-HiBiT-HBx plasmid. II Fast Mutagenesis Kit V2 (C214, Novezan, China) Instructions for use of the experimental procedure successfully replaced the N-terminal portion of the HBx sequence in the recombinant cccDNA plasmid with the coding sequence of the HiBiT tag. Figure 1 As shown in A. Sanger sequencing results confirmed the successful construction of the plasmid. The specific plasmid construction process is as follows:

[0062] 1. Using the prcccDNA plasmid as a template, use the Phanta Max Super-Fidelity DNA Polymerase and primers HiBiT-SF (SEQ ID NO.1) and HiBiT-SR (SEQ ID NO.2) in the kit to amplify the target plasmid. The reaction system is:

[0063]

[0064] The PCR reaction conditions are as follows:

[0065]

[0066] 2. Digestion: After the reaction is completed, a small amount of amplified product is taken for agarose gel electrophoresis detection. The amplified band is specific, and then the amplified product is digested with Dpn I to remove the methylated template plasmid. The reaction system is: Dpn I 1μl, amplified product 49μl. The reaction conditions are: constant temperature reaction at 37℃ for 2h to obtain Dpn I digestion product.

[0067] 3. Recombination: Subsequently, the Dpn I digestion product can be efficiently recombined at the site to be mutated under the catalysis of Exnase II to achieve in vitro circularization of linear DNA. The reaction system is as follows:

[0068] DpnI digestion products 108ng 5×CEIIBuffer 4μl ExnaseII 2μl <![CDATA[ddH2O]]> Add to 20 μl

[0069] The reaction conditions are: 37°C, 30 min; cool to 4°C or immediately cool on ice.

[0070] 4. Transformation: The recombinant product was transformed into TOP10 competent bacteria, plated, cloned and screened, sequenced and identified, and the correct clone was named prcccDNA-HiBiT-HBx( Figure 1 A is its vector map), prcccDNA-HiBiT-HBx is a plasmid in which the first 33 bp of the HBx N-terminus in the plasmid prcccDNA is replaced with the HiBiT sequence; rcccDNA-HiBiT-HBx expressed by prcccDNA-HiBiT-HBx is a plasmid in which the first 33 bp of the HBx N-terminus in the rcccDNA is replaced with the HiBiT sequence ( Figure 1 B is its structural diagram).

[0071] 2. Functional identification of recombinant plasmid prcccDNA-HiBiT-HBx

[0072] NanoBiT is a two-component structural complementary system consisting of two subunits, HighNanoBiT (HiBiT) and Large NanoBiT (LgBiT). The two individual subunits do not have enzymatic activity, but when HiBiT and LgBiT bind in cells or in vitro, the complex restores its nanoluciferase activity. The HiBiT tag is only 11 amino acids long and acts like a bright luciferase, allowing simple and sensitive quantification of tagged proteins within a linear dynamic range of 7 orders of magnitude. Because of its wide and sensitive dynamic range, the HiBiT reporter gene was used to monitor HBx expression and protein levels in the present study.

[0073] Cre (Cyclization Recombination Enzyme) is a recombinase derived from P1 phage. The gene coding region sequence is 1029 bp in length, and is a 38 kDa polypeptide monomer protein composed of 343 amino acids. The C-terminal domain of Cre recombinase contains a catalytic active site that can catalyze the recombination between specific sites in the DNA molecule. At the same time, Cre can also recognize specific DNA sequences, namely loxP sites, so that gene recombination occurs between two loxP sites. Locus of X-over P1 (LoxP) is a 34 bp sequence located in P1 phage, which is composed of two 13 bp reverse palindromic sequences and an 8 bp intermediate spacer sequence. The reverse palindromic sequence is the recognition and binding region of Cre recombinase, and the spacer sequence determines the direction of the LoxP sequence.

[0074] To verify whether HiBiT tag can play a role in characterizing HBx protein expression in cells, we transiently co-transfected prcccDNA-HiBiT-HBx and pCMV-Cre plasmids at a 1:1 ratio using Lipofectamine 3000 transfection reagent in HepG2 and Huh7 cell lines. The experimental operation steps in the HiBiT detection kit manual were used to detect the activity level of HiBiT. The experimental results showed that after 72 hours of co-transfection of prcccDNA-HiBiT-HBx and pCMV-Cre plasmids in HepG2 cells and Huh7 cells, the expression of HiBiT protein levels could be achieved, which to some extent characterized the expression level of HBx protein and played the role of reporter gene ( Figure 2 B).

[0075] To verify that this model can form cccDNA in cells, we transiently co-transfected prcccDNA-HiBiT-HBx and pCMV-Cre plasmids in HepG2 and Huh7 cell lines with Lipofectamine 3000 transfection reagent at a ratio of 1:1. 72 hours after plasmid transfection, cccDNA was extracted by Hirt method and the expression of cccDNA was detected by Southern blotting. The results showed that prcccDNA-HiBiT-HBx, like prcccDNA, can also express cccDNA in the cell model through Cre recombinase ( Figure 2 C).

[0076] To verify that this model can form minichromosomes in cells, we transiently co-transfected prcccDNA-HiBiT-HBx and pCMV-Cre plasmids in a 1:1 ratio using Lipofectamine3000 transfection reagent in the Huh7 cell line. 72 hours after plasmid transfection, the cells were fixed with formaldehyde and the formation of minichromosomes was detected by CHIP-qPCR. The specific process is as follows:

[0077] 1. Crosslink the DNA and target protein on the chromatin with formaldehyde: digest and harvest the cells with trypsin substitute, resuspend in 1 mL of 37°C preheated culture medium and add 4 mL of culture medium. Add 135 μl of 36.5% formaldehyde solution to 5 mL of sample and quickly invert to mix. Incubate at room temperature for 10 min. Add 570 μl of 1.25M Glycine (Shanghai Shenggong Biotechnology, China) solution and incubate at room temperature for 5 min. Centrifuge at 4°C, 470g for 10 min, discard the supernatant, add 5 ml of precooled 1× PBS to resuspend the cells, centrifuge at 4°C, 470g for 10 min, discard the supernatant. Resuspend the cells with 1 ml of precooled 1× PBS, place in a 1.5 ml centrifuge tube, centrifuge at 4°C, 14800 rpm for 5 min, discard the supernatant, centrifuge again for 30 s, aspirate the supernatant, and quickly place in liquid nitrogen.

[0078] 2. Fragment the chromatin by ultrasound or enzymatic hydrolysis: Add 1.25 ml SDS lysis buffer to the cells fixed with formaldehyde and place at room temperature for 5 minutes. In an ultrasonic instrument, at 30% power, 5 times, ultrasonically fragment the chromatin to a size of about 200-300 bp.

[0079] 3. Chromatin fragments containing the target protein are enriched and purified by antibodies: After ultrasound, centrifuge at 25°C, 14800rpm for 10min, add the supernatant to a 15mL Ultracel-50K (Millipore) column, and centrifuge at 25°C, 4000g for 5min. Discard the filtrate, add 1mL IP buffer, centrifuge at 25°C, 4000g for 5min, and repeat this step once. Transfer the remaining supernatant to a 1.5mL centrifuge tube, wash the Ultracel-50K (Millipore) column 4 times with 200μl IP buffer and transfer to a 1.5mL centrifuge tube. Take 100μl as input. Take 500μl and add 150μl Detergent Mix, then add the corresponding antibodies respectively, and incubate at 4°C with rotation overnight. The next day, wash the magnetic beads 3 times with Blocking buffer, 1min each time. Resuspend the magnetic beads with 50μl Blocking buffer, add to the chromatin incubated overnight, incubate at 4℃ with rotation for 2h, then add 500μl pre-cooled RIPA buffer to wash the magnetic beads 5 times, 1min each time. Add 500μl room temperature TE buffer to wash the magnetic beads once. Discard the TE buffer and centrifuge at 3000rpm for 3min at room temperature. Use a 10μl gun to suck out the remaining liquid. Add 100μl Elutionbuffer and transfer to a new 1.5mL centrifuge tube, vortex for 10s, and oscillate in a constant temperature oscillator at 65℃ and 1500rpm for 15min. Centrifuge at maximum speed at room temperature for 30s and transfer the supernatant (i.e., ChIP product) to a new 1.5mL centrifuge tube.

[0080] 4. Remove the cross-linking between DNA and target protein by heating: Place the ChIP product and Input product in a 65℃ water bath overnight to remove the cross-linking.

[0081] 5. Extract genome: Add TE buffer to balance the volume of ChIP product and Input to 400μl, add 10μl RNaseA, and digest in a 37℃ water bath for 1h. Add 25μl Proteinase K Mix (15μl 5M NaCl, 2μl 10mg / mL Glycogen, 8μl PK enzyme) and incubate in a 60℃ water bath for 1h. Add 440μl phenol chloroform, vortex for 10s, and centrifuge at 14800 rpm at room temperature for 8min. Take 400μl supernatant, add 800μl pre-cooled anhydrous ethanol, and vortex for 10s. Incubate in a -80℃ refrigerator for 30min, then centrifuge at 4℃, 14000rpm for 15min. Discard the supernatant, add pre-cooled 75% anhydrous ethanol, and centrifuge at 4℃, 14000rpm for 15min. Discard the supernatant, open the lid and let it dry for about 5 minutes. When the precipitate becomes transparent, add 20 μl ddH2O and dissolve it in a 37°C water bath for 15 minutes.

[0082] 6. CHIP-qPCR: Use the genomic product obtained in the above step as a template and P3 (SEQ ID NO.3) and P4 (SEQ ID NO.4) as primers for qPCR.

[0083] The reaction system is as follows:

[0084] <![CDATA[HotStart TM 2X SYBR Green qPCR Master Mix]]> 5μl Genome products 1μl P3 (7.2 μM) 0.5μl P4 (7.2 μM) 0.5μl ddH2O 3μl

[0085] The reaction conditions are as follows:

[0086]

[0087] The results showed that prcccDNA-HiBiT-HBx, like prcccDNA, could also form minichromosomes in cell models through Cre recombinase ( Figure 2 D). The basic principle of ChIP is that when treated with formaldehyde in living cells, covalent bonds will be generated between proteins and proteins, and proteins and nucleic acids (DNA or RNA) that are close to each other. In cells, when TF and promoter bind to each other, they must be close to each other or fit together. At this time, treatment with formaldehyde can generate covalent bonds between them. The fixed protein-DNA complex is randomly cut into small chromatin fragments within a certain length range by ultrasound or enzyme treatment, and then the complex is precipitated through the specific recognition reaction of antigen and antibody, specifically enriching the DNA fragments bound by the target protein, and obtaining information on the interaction between protein and DNA by purifying and detecting the target fragments. Figure 2 The results in D showed that there was interaction between cccDNA and histones, indicating the formation of minichromosomes.

[0088] Example 2: Antiviral drug screening

[0089] Our laboratory screened out some small molecule compounds in preliminary experiments, and ordered their derivatives based on the experimental results for further screening using the above-mentioned cell model.

[0090] The principle of the cell model of the present invention is: the loxP chimeric intron is designed into the monomeric HBV genome in the precursor plasmid (prcccDNA), and then it is excised using Cre / loxP-mediated DNA recombination and enters the 3.3kbrcccDNA in the hepatocyte nucleus. The chimeric intron is spliced ​​from the RNA transcript without interrupting the HBV life cycle. In cultured liver cancer cells, co-transfection of prcccDNA and Cre plasmid leads to the accumulation of nuclear rcccDNA, which is epigenetically organized into minichromosomes. The rcccDNA contains the HBx sequence, which is labeled with a HiBiT tag, and the state of the minichromosome can be characterized by detecting HiBiT activity.

[0091] The schematic diagram of the screening process of antiviral drugs using the cell model of the present invention is as follows Figure 3 As shown in A, the specific process is:

[0092] In order to convert as much prcccDNA as possible into rcccDNA, we transiently co-transfected prcccDNA-HiBiT-HBx and pCMV-Cre plasmids at a ratio of 1:19 in HepG2 cell lines using Lipofectamine3000 transfection reagent.

[0093] 24 hours after transfection, the cells in the 6 cm culture dish were digested, plated in a 12-well plate, and treated with small molecule compounds. At the same time, 2% DMSO was added to the culture medium to prevent the loss and dilution of cccDNA caused by cell division. The experimental operation steps of the HiBiT detection kit instructions are used to detect the activity level of HiBiT ( Figure 3 B); RT-qPCR was performed to detect HBV pgRNA levels by extracting cell RNA using the Trizol method ( Figure 3 C).

[0094] The reaction system is as follows:

[0095] <![CDATA[HotStart TM 2X SYBR Green qPCR Master Mix]]> 5μl cDNA 1μl HBV pgRNA-F (7.2 μM) 0.5μl HBV pgRNA-R (7.2μM) 0.5μl ddH2O 3μl

[0096] The reaction conditions are as follows:

[0097]

[0098]

[0099] The results show that Figure 3), in the HepG2 cell line, among the 20 derivatives, compounds 3, 4, 9, 10, 15, and 20 all had a certain inhibitory effect on the activity level of HiBiT and the transcriptional activity of HBV, among which compounds 3 and 10 had the most significant effects, but compound 3 was difficult to dissolve, so we finally selected compound 10 for subsequent experiments.

[0100] Example 3: Evaluation of Screened Drugs by HBV Cell Infection Model

[0101] Taking into account the HiBiT activity level and the degree of decrease in HBVpgRNA level, solubility, CC50 and other factors, according to the results in Example 2, we finally selected compound 10 for subsequent experiments. Compound 10 is 5-(2,3-dihydro-1H-inden-5-yl)-2-(thiophen-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine, CAS number: 883007-39-4, chemical formula: C 20 H 14 F3N3S, whose chemical structure is shown in Formula I:

[0102]

[0103] We treated HBV-infected HepG2-NTCP with compound 10: HepG2-NTCP cells were seeded in 12-well plates with 280,000 cells per well. After 24 hours, the medium was replaced with PMM medium, and the cells were treated with different concentration gradients (0, 5, 10 μM) of compound 10 for 24 hours. HBV virus was diluted in infection medium containing 4% PEG8000 (1000 HBV viruses were required for each cell), then added to 12-well plates, and the cells were treated with different concentration gradients (0, 5, 10 μM) of compound 10 for 24 hours. The cells were rinsed 3 times with PBS buffer, the infection medium was replaced, and the medium was replaced every other day. Each time the medium was replaced, the cells were treated with different concentration gradients (0, 5, 10 μM) of compound 10.

[0104] On the seventh day, the HBeAg level in the culture supernatant was detected by ELISA test using the HBeAg antigen kit ( Figure 4 C); Trizol method was used to extract cell RNA and RT-qPCR and Northern blot experiments were performed to detect HBV RNAs levels ( Figure 4 D, E, F); Cell proteins were extracted by 1×SDS-PAGE loading buffer for Western blot experiments, and then the expression level of HBc protein was detected using anti-HBc antibody ( Figure 4 G). The results showed that after treatment with compound 10, the HBeAg level, HBV RNAs level and HBc protein expression level were significantly decreased.

[0105] Compare compound 10 in the present invention with compound C screened in the previous stage of this laboratory 19 H 14 The results of CC50 (half toxic concentration of cells) and IC50 (half inhibitory concentration) of F3N3O2S (see Chinese patent application publication number CN117679420A) are as follows: Figure 5 As shown: In HepG2-NTCP cells, the CC50 of compound 10 is 961.3 μM, and that of compound C is 19 H 14 CC50 of F3N3O2S = 230.1 μM ( Figure 5 A); In HepAD38 cells, the CC50 of compound 10 is 513.3 μM, and that of compound C is 513.3 μM. 19 H 14 CC50 of F3N3O2S = 181.7 μM ( Figure 5 B); In HepG2 cells, the CC50 of compound 10 is 641.8 μM, and that of compound C is 641.8 μM. 19 H 14 CC50 of F3N3O2S = 140.2 μM ( Figure 5 C); In Huh7 cells, the CC50 of compound 10 is 117.4 μM, and the CC50 of compound C is 117.4 μM. 19 H 14 CC50 of F3N3O2S = 105.1 μM ( Figure 5 D); In HepG2-NTCP cells, the IC50 of compound 10 is 1.593 μM, and that of compound C is 1.593 μM. 19 H 14 IC50 of F3N3O2S = 10.9 μM ( Figure 5 E). In summary, the effect of compound 10 is better than that of compound C. 19 H 14 F3N3O2S has better effects and better prospects for clinical application.

Claims

1. A recombinant plasmid prcccDNA-HiBiT-HBx, characterized in that: The backbone vector is the plasmid prcccDNA, and the recombinant plasmid prcccDNA-HiBiT-HBx is obtained by replacing the first 33 bp of the N-terminus of the HBx sequence of the prcccDNA plasmid with the HiBiT tag sequence.

2. The recombinant plasmid prcccDNA-HiBiT-HBx according to claim 1, characterized in that: The first 33 bp sequence at the N-terminus of the HBx sequence is: 5'-TGTTGTCCTATCCCGCAAATATACATCGTTTCC-3'; The HiBiT tag sequence is: 5'-ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATCAGC-3'.

3. The method for constructing the recombinant plasmid prcccDNA-HiBiT-HBx according to claim 1 or 2, characterized in that: The method comprises the following steps: using prcccDNA plasmid as a template, performing PCR amplification on HiBiT-SF / HiBiT-SR using primers shown in SEQ ID NO.1 and 2, digesting the amplified product with Dpn I, efficiently recombining the digested product with a recombinase to realize in vitro circularization of linear DNA, transforming the recombinant product into competent bacteria, picking a single clone, and sequencing the correct clone as the recombinant plasmid prcccDNA-HiBiT-HBx; preferably, the recombinase is Exnase II.

4. A minichromosome state characterization cell model for drug screening, characterized in that: The prcccDNA-HiBiT-HBx described in claim 1 or 2 is co-transfected with plasmid Cre into a liver cell line to obtain transfected cells.

5. Use of the minichromosome state characterization cell model according to claim 4 in screening drugs against hepatitis B virus.

6. The use according to claim 5, characterized in that: Co-transfect the prcccDNA-HiBiT-HBx and Cre plasmid of claim 1 or 2 into a liver cell line, digest the transfected cells, and then treat them with the selected compound. After the treatment, detect the HiBiT activity level; preferably, use HiBiT detection kit was used to detect the activity level of HiBiT.

7. Use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of HBV inhibitors or drugs for treating hepatitis B virus infectious diseases or conditions:

8. The use according to claim 1, characterized in that: The compound represented by formula I or a pharmaceutically acceptable salt thereof inhibits the transcription of hepatitis B virus; Preferably, the compound represented by formula I or a pharmaceutically acceptable salt thereof reduces the expression levels of HBV total RNAs, HBeAg, HBV pgRNA and HBV nucleoprotein.

9. A pharmaceutical composition for inhibiting HBV replication or treating hepatitis B virus infectious diseases or symptoms, characterized in that: Contains the compound represented by formula I in claim 7 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9 is characterized in that: The product dosage form of the pharmaceutical composition is capsule, tablet, pill, paste, granule, oral solution preparation, microcapsule preparation or injection.

Images