WO2013113242A1 - 抗hbv的化合物的筛选方法 - Google Patents

抗hbv的化合物的筛选方法 Download PDF

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WO2013113242A1
WO2013113242A1 PCT/CN2012/087546 CN2012087546W WO2013113242A1 WO 2013113242 A1 WO2013113242 A1 WO 2013113242A1 CN 2012087546 W CN2012087546 W CN 2012087546W WO 2013113242 A1 WO2013113242 A1 WO 2013113242A1
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hbv
core protein
screening
cells
compound
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French (fr)
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左建平
南发俊
杨莉
童贤昆
张仰明
王桂凤
唐炜
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中国科学院上海药物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

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  • the present invention relates to the field of molecular biology and biomedical technology, and in particular, the present invention relates to a medicament for targeting a C-terminal amino acid sequence of a hepatitis B virus (HBV) core protein as a drug target site.
  • Screening method Background Art Chronic hepatitis B infection is a global infectious disease that seriously endangers human health. In China, liver disease caused by hepatitis B virus ranks among the top ten causes of death in China, which seriously affects people's normal work and life.
  • the listed small molecule anti-hepatitis B virus drugs are mainly nucleoside drugs, such as lamivudine, entecavir, etc., although these drugs are effective in inhibiting viral replication, but due to the existing nucleoside anti-hepatitis B Viral drugs have a single target (HBV DNA polymerase), and long-term use of existing resistance mutations. Therefore, it is an important and urgent task to find new targets different from the mechanism of action of nucleoside anti-HBV drugs, and to develop new drugs for the treatment of hepatitis B, especially non-nucleoside small molecule anti-HBV drugs.
  • nucleoside drugs such as lamivudine, entecavir, etc.
  • the non-nucleoside small molecule anti-HBV active compounds have been reported to be: Heteroaromatic Dihydropyrimidine (HAP) series compounds, representative compound BAY38-7690, which interfere with the core protein by interacting with the 113-143 amino acid residues of the core protein.
  • HAP Heteroaromatic Dihydropyrimidine
  • BAY38-7690 Representative compound BAY38-7690, which interfere with the core protein by interacting with the 113-143 amino acid residues of the core protein.
  • the core protein monomer is over-accumulated and degraded in the cell; the hydrophobic fluorescent probe bis-ANS can bind to the core protein to form a non-encapsid macromer, misleading the normal of the core protein.
  • phenylacrylamides AT-61 and AT-130 can promote the assembly rate of core proteins, making pgRNA less than packaged, thus reducing the level of HBV-DNA replication; 8-1 specifically interferes with the interaction of nuclear transcription factors of host cells with the HBV promoter and down-regulates the transcription level of viral RNA.
  • the HBV core protein has a total of 34 amino acids at the C-terminus, which is a protamine-like structure. Among them, there are 16 arginine, which form 3 SPRRR sequences and 4 arginine accumulation regions, which are highly basic ends and can bind to the pre-genome.
  • the RNA and polymerase complex (pgRNA-Pol) initiates nuclear shell packaging. Currently It has not been reported that the core protein C-terminal amino acid is used as a drug target to screen anti-HBV drugs.
  • Recombinant DNA technology is a biotechnology commonly used in molecular biology. It refers to the splicing and recombination of a gene (a donor) gene and a vector in vitro, and then transferred to another organism (receptor).
  • a DNA in vitro procedure that is stably inherited and expresses new products or new traits according to people's wishes, also known as molecular cloning techniques.
  • the present invention constructs a variety of eukaryotic expression plasmids by recombinant DNA technology to express a C-terminal deletion, truncation or mutation of the core protein, and determines four positively charged arginine-rich regions at the C-terminus of the core protein (RRRDRGR, SPRRR, SPRRRR).
  • the present invention provides a screening method for a compound resistant to HBV, wherein the method uses a C-terminal amino acid sequence of a HBV core protein as a drug target to screen for an anti-HBV compound, the HBV core protein C
  • the terminal amino acid sequence comprises at least STLPETTVVRRRDRGR.
  • the C-terminal amino acid sequence of the HBV core protein may be:
  • the method includes the following steps:
  • An eukaryotic expression plasmid comprising the C-terminal amino acid sequence of HBV core protein is constructed by PCR, wherein the HBV core protein C-terminal amino acid sequence comprises at least STLPETTVVRRRDRGR.
  • the plasmid obtained in the step (a) is transiently transfected into a liver cancer cell (Huh7 cell, HepG2 cell or the like, preferably Huh7 cell). After 4 to 6 hours, the screening compound is administered for 48 to 96 hours.
  • a liver cancer cell Human H7 cell, HepG2 cell or the like, preferably Huh7 cell.
  • the screening compound is administered for 48 to 96 hours.
  • Cells were collected after drug treatment, and non-denaturing agarose gel electrophoresis was used to screen for compounds that induce packaging of abnormally empty HBV core particles.
  • the screening compound in the step (b) is treated by: co-culturing the transfected liver cancer cells or the screening compound, and the concentration of the compound can be set as needed.
  • the step (c) is specifically as follows: the cells are collected after treatment with the screening compound, and the cell lysate is sampled in a 1.8% agarose gel well and electrophoresed at 4 ° C for 2 to 3 hours, preferably 2.5 hours. After the end of the electrophoresis, the membrane was transferred by siphon overnight. The resulting sample can be transferred to a nitrocellulose membrane, and an abnormal core particle induced by the screening compound is detected by an anti-HBc antibody (Abeam) by a method similar to Western blot.
  • the abnormal core particle refers to an empty HBV core particle free of HBV virus genetic material (viral nucleic acid), which can hinder the further infection replication of the HBV virus.
  • the method for screening compounds provided by the present invention can be used for screening and discovering abnormally packaged anti-HBV replication active compounds for inducing HBV core particles, in order to find that compounds act on the C-terminal target sequence of HBV core protein, and induce core protein assembly to be free of HBV virus inheritance.
  • the empty (HBV) core of the substance (viral nucleic acid) blocks the further replication of the HBV virus.
  • the screening methods of the compounds provided by the present invention can also be used to detect the activity of novel antiviral drugs for treating hepatitis B for these target sites which are obtained in other ways.
  • Fig. 1 is a gel electrophoresis pattern of core particles obtained by transfecting Huh7 cells (Example 6) constructed in Example 1 according to the present invention.
  • Fig. 2 is a gel electrophoresis pattern of the core particles obtained by transfecting Huh7 cells (Example 6) in accordance with Example 2 constructed in accordance with the present invention.
  • Fig. 3 is a gel electrophoresis pattern of the core particles obtained by transfecting the Huh7 cells (Example 6) of the plasmid constructed in Example 3 of the present invention.
  • Fig. 4 is a gel diagram of a core particle gel obtained by transfecting Huh7 cells (Example 6) of plasmids constructed in Examples 4 and 5 of the present invention.
  • Figure 5 is a gel electrophoresis pattern of core particles obtained according to Example 7 of the present invention.
  • Fig. 6 is a gel electrophoresis pattern of HBV DNA obtained in Example 8 according to the present invention.
  • Figure 7 is a graph showing the results of real-time quantitative PCR obtained according to Example 9 of the present invention.
  • Figure 8 is a graph showing the results of real-time quantitative PCR obtained according to Example 10 of the present invention.
  • Figure 9 is a diagram showing a PCR reaction procedure according to Example 1 of the present invention.
  • Figure 10 is a diagram showing the PCR reaction procedure of Example 2 according to the present invention.
  • FIG 11 is a diagram showing the PCR reaction procedure of Example 9 according to the present invention.
  • BEST MODE FOR CARRYING OUT THE INVENTION The following examples are intended to facilitate the understanding of the present invention by those skilled in the art, but are not intended to limit the invention in any way.
  • Example 1 Construction of a full-length HBV core protein eukaryotic expression plasmid targeting the target sequence HBc-1 pHBcl85
  • the cDNA fragment encoding the full-length core protein and the pcDNA3.1 plasmid (purchased from Invitrogen) were cloned and expressed into the recipient strain DH5a (purchased from the day) by expressing 1.3-fold full-length HBV genome (adw subtype) pHBV1.3 as a template.
  • the eukaryotic expression plasmid pHBcl85 of the full-length core protein was obtained by screening and identification.
  • the amplification primers are as follows:
  • the plasmid pHBcl85 expresses the full-length HBV core protein in eukaryotic cells, and its C-terminus includes four arginine-rich regions, three of which are SPRRR repeats and the sequence is HBc-1 (SEQ ID NO: 1):
  • the vector fragment pHBCTD was generated by PCR, and the amplification primers were as follows:
  • the synthetic fragment was inserted into the above-mentioned vector fragment pHBCTD generated by PCR and transformed into E. coli receptor bacteria (same as in Example 1) by homologous recombination technique. After screening and identification, the C-terminal truncated core protein eukaryotic was obtained. Expression plasmid pHBcl71.
  • the PCR reaction procedure is shown in Figure 10.
  • the homologous recombination reaction conditions are as follows:
  • the conversion conditions were the same as in Example 1.
  • the plasmid pHBc l expresses a C-terminal partially deleted core protein in eukaryotic cells, and its C-terminus includes two arginine-rich regions, and two SPRRR repeats are deleted, and the sequence is HBc-2 (SEQ ID NO: 2). :
  • Example 3 Construction of the C-terminal truncation of the target sequence HBc-3 HBV core protein eukaryotic expression plasmid pHBcl63
  • the synthetic fragment was inserted into the above-mentioned vector fragment pHBCTD generated by PCR and transformed into E. coli receptor bacteria (same as in Example 1) by homologous recombination technique. After screening and identification, the C-terminal truncated core protein eukaryotic expression was obtained. Expression plasmid pHBcl63.
  • the plasmid pHBcl63 expresses a C-terminal partially deleted core protein in eukaryotic cells, and its C-terminus includes two arginine-rich regions, and two SPRRR repeats are deleted, and the sequence is HBc-3 (SEQ ID NO: 3):
  • a DNA insert encoding the C-terminal 141-156 amino acid of the HBV core protein was synthesized as follows (SEQ ID NO: 12):
  • the synthetic fragment was inserted into the vector fragment pHBCTD generated by PCR in Example 2 by homologous recombination technique, and transformed into E. coli recipient bacteria (same as in Example 1). After screening and identification, a C-terminal truncated core was obtained.
  • the plasmid pHBcl56 expresses a core protein deleted at the C-terminal portion in eukaryotic cells, and has only one arginine-rich region at the C-terminus, and three SPRRR repeats are deleted, and the sequence is HBc-4 (SEQ ID NO: 4):
  • Example 5 Construction of a C-terminal deletion HBV core protein eukaryotic expression plasmid pHBcl56M A DNA insert encoding the amino acid at positions C-141-156 of the HBV core protein was synthesized, and arginine at 150, 151, 152, 154 and 156 was Alanine substitution, the sequence is as follows (SEQ ID NO:
  • the synthetic fragment was inserted into the vector pHBCTD by homologous recombination technology and transformed into E. coli recipient strain (same as in Example 1). After screening and identification, the C-terminal truncated HBV core protein eukaryotic expression plasmid pHBcl56M was obtained.
  • the plasmid pHBcl56M expresses a C-terminally mutated core protein in eukaryotic cells, and has no arginine-rich region at the C-terminus.
  • the sequence is:
  • Huh 7 cells human hepatoma cell line, submitted by Wuhan Virus of Chinese Academy of Sciences, preserved in our laboratory
  • Huh 7 cells were cultured in DMEM containing 10% FBS, and cultured at 5% C0 2 37 °C.
  • Huh 7 cells were seeded in 6-well plates at a density of 5 x 105 cells/well for overnight adherence. Times Transfection experiments were carried out according to Lipofectamine 2000 Transfection Reagent (Invitrogen). The plasmid was transfected with the corresponding eukaryotic expression plasmids prepared in the above Examples 1, 2, 3, 4 and 5.
  • the present invention selects the anti-hepatitis B virus drug isothiazide (NZ-4) synthesized by the present invention according to the prior art as a test compound (the final concentration is 20 micromolar, 10 micromolar or 5 ⁇ mol of DMEM medium was cultured. After 48 hours of culture, the cells were used for the corresponding detection.
  • Example 7 Screening of active compounds against HBV by non-denaturing agarose gel electrophoresis
  • HuhG2.2.15 cells (sponsored by Wuhan Virus of Chinese Academy of Sciences, preserved in our laboratory) or Huh 7 cells transiently transfected with the plasmid prepared in Example 6 were treated with different concentrations of test compounds (ie, the final concentration was 20 ⁇ mol, 10 ⁇ m).
  • the cells were collected, and the cell lysate was centrifuged to remove nuclei and cell debris, and then loaded into a 1.8% agarose gel well and electrophoresed at 4 ° C for 2.5 hours. After the end of the electrophoresis, the membrane was transferred by siphon overnight.
  • the sample can be transferred to a nitrocellulose membrane and the core particles are detected using an anti-HBc antibody (Abeam) in a manner similar to Western blot.
  • Gel in situ denaturation can also be carried out with denaturing solution (1.5 M NaCl/0.5 M NaOH) for 45 min after the end of electrophoresis, neutralized solution (1.5 M NaCl/0.5 M Tris CI, pH 7.0) for 45 min, and then transferred.
  • denaturing solution 1.5 M NaCl/0.5 M NaOH
  • neutralized solution 1.5 M NaCl/0.5 M Tris CI, pH 7.0
  • the subsequent method was similar to Southern blot and Northern blot, and the nucleic acid level in the nucleocapsid was detected using a HBV-specific probe (Daan Gene).
  • HepG2.2.15 cells are derived from human hepatoma cell line HepG2 cells, which can stably transfect HBV gene, stably perform HBV genome replication, and stably express HBV, and can verify the anti-HBV activity of the compounds selected by the method. It can also be used to detect the effects of compounds on HBV DNA.
  • the core particle gel electrophoresis obtained by transfecting the plasmid pHBcl85 constructed in the above Example 1 with Huh7 cells for compound screening is shown in Fig. 1.
  • the experimental results show that the compound NZ-4 obtained by screening can bind to the C-terminal of the core protein.
  • the arginine-rich region induces abnormal core particle formation, which is independent of the involvement of pgRNA.
  • the gel electrophoresis pattern of the core particle obtained by transfecting the plasmid pHBc1 constructed in the above Example 2 into the compound for screening is shown in Fig. 2.
  • the experimental results show that NZ-4 can induce the abnormal formation of the C-terminally truncated core protein.
  • the gel electrophoresis pattern of the core particle obtained by transfecting the plasmid pHBcl63 constructed in the above Example 3 into the compound for screening of the compound is shown in Fig. 3.
  • the result of the test indicates that the NZ-4 can induce the C-terminally truncated core protein to form an abnormal core. Particles.
  • the gel electrophoresis patterns of the core particles obtained by screening the compounds pHBcl56 and pHBcl56M constructed in the above Examples 4 and 5 for compound screening are shown in Fig. 4.
  • the results of the experiment indicate that the NZ-4 mutation to the C-terminal arginine is The core protein of alanine is ineffective, indicating that the positively charged arginine in this region is a key target for drug screening.
  • the abnormal core particles induced by NZ-4 on HepG2.2.15 cells were empty core particles, free of viral nucleic acids, and the DNA level in the nucleocapsid was significantly reduced.
  • GAPDH is glyceraldehyde-3-phosphate dehydrogenase, and its gene is a housekeeping gene, which is expressed at high levels in almost all cells.
  • the amount of GAPDH protein expression in the same species is generally constant and is therefore used as a standardized internal reference for experimental manipulation in this example.
  • Example 8 Southern blotting Determination of compound anti-HBV activity HepG2.2.15 cell lysate was centrifuged to remove nuclei and cell debris, nuclease cleavage, proteinase K digestion, phenol chloroform extraction, isopropanol precipitation Get viral DNA. The DNA was loaded into a 0.8% agarose gel well and electrophoresed at 70 V for 1 hour. After electrophoresis, the gel was in situ denatured with denaturing solution (1.5 M NaCl/0.5 M NaOH) for 45 min, neutralized solution (1.5 M NaCl/0.5 M Tris CI, H 7.0) for 45 min, and transferred to Hybond-N+.
  • denaturing solution 1.5 M NaCl/0.5 M NaOH
  • neutralized solution 1.5 M NaCl/0.5 M Tris CI, H 7.0
  • Fig. 6 shows a gel electrophoresis pattern of the obtained HBV DNA, which shows that NZ-4 inhibits three replication intermediates of the virus on HepG2.2.15 cells, and reduces intracellular HBV-DNA levels.
  • Example 9 Real-time RPCR for detecting the number of virus particles in cell culture supernatants
  • Figure 7 shows a graph of the obtained real-time PCR results, which showed that NZ-4 reduced the level of virions in the HepG2.2.15 cell culture supernatant.
  • Example 10 HBV transgenic mouse model (Balb/c background)
  • 6-week-old HBV transgenic mice purchased from the 458th Hospital of the People's Liberation Army of Guangzhou, Institute of Liver Diseases
  • model group prescription adjuvant treatment group
  • oral (gavage) administration 6-7 mice in each group were treated continuously for 28 days, before administration (0d), 7th day after administration (7d), 14th day (14d), 21st day (21d) and On the 28th day (28d), blood was taken from the fundus venous plexus, and the serum of the mice was collected, and the HBV DNA was extracted for real-time PCR detection (the same as in Example 9).
  • Figure 8 shows the obtained real-time PCR results, showing that NZ-4 can significantly reduce HBV DNA levels in the serum of HBV transgenic mice.
  • the present invention uses a plasmid pHBcl85 to transiently transfect Huh7 cells to express a full-length core protein, and a non-denaturing agarose gel electrophoresis method for screening a compound capable of inducing an abnormally empty core particle package, and determining 45 C-terminal ends of the HBV core protein.
  • the amino acid (aal41-aal85, adw subtype) is a target sequence for screening for antiviral drugs, and its sequence is SEQ ID NO: l:
  • the present invention expresses a C-terminal truncated core protein by transiently transfecting Huh7 cells with a plasmid pHBc1, and screens a compound capable of inducing abnormal core particle packaging by non-denaturing agarose gel electrophoresis to determine a core protein.
  • the C-terminal truncation sequence (aal41-aal71) is a target sequence for antiviral drug screening, the sequence of which is SEQ ID NO: 2:
  • the present invention expresses a C-terminal truncated core protein by transiently transfecting Huh7 cells with a plasmid pHBcl63, and screens a compound capable of inducing abnormal core particle packaging by non-denaturing agarose gel electrophoresis, and determines a C-terminal truncation of the core protein.
  • the sequence (aal41-aal63) is a target sequence for screening for antiviral drugs, the sequence of which is SEQ ID NO: 3:
  • the present invention expresses a C-terminal truncated core protein by transient transfection of Huh7 cells with a plasmid pHBcl56, and screens a compound capable of inducing abnormal core particle packaging by non-denaturing agarose gel electrophoresis, and determines a C-terminal fragment of the core protein.
  • the short sequence (aal41-aal56) is a target sequence for screening for antiviral drugs, the sequence of which is SEQ ID NO: 4:
  • an arginine-rich region (aal50- aa 156) at the C-terminus of the core protein of the present invention is a target sequence for antiviral drug screening, but if 5 of the arginine are mutated to alanine, The C-terminal mutant core protein no longer has the activity of screening for anti-HBV compounds (as described in the plasmid pHBcl56 and pHBcl56M in Example 7), the sequence of the arginine-rich region is Arg Arg Arg Asp Arg Gly Arg
  • Table 1 shows the core protein C-terminal target sequences of HBV (adw subtype) determined according to the above examples, which correspond to Examples 1, 2, 3 and 4 in order from top to bottom. Table 1
  • Sequence target sequence target sequence length position number (aa) (aa)
  • Example 11 Detection of anti-HBV activity of non-nucleoside small molecule compound isothiazide (NZ-4)
  • NZ- non-nucleoside small molecule compound isothiazide
  • the main detection steps are as follows:
  • HepG2.2.15 cells were treated with a final concentration of 20 ⁇ mol, 10 ⁇ mol and 5 ⁇ mol of isothiazolidine (NZ-4) for 24 days after adherence for 24 hours.
  • the cell lysate was applied to a 1.8% agarose gel well and electrophoresed at 4 ° C for 2.5 hours. After the end of the electrophoresis, the membrane was transferred by siphon overnight. The sample can be transferred to a nitrocellulose membrane, and an abnormal core particle induced by the screening compound is detected by an anti-HBc antibody (Abeam) using a method similar to Western blot. At the same time, the same sample was prepared for gel electrophoresis. After electrophoresis, the gel was in situ denatured with a denaturing solution (1.5 M NaCl/0.5 M NaOH) for 45 min, neutralizing solution (1.5 M NaCl/0.5 M Tris CI, pH 7.0).
  • a denaturing solution 1.5 M NaCl/0.5 M NaOH
  • neutralizing solution 1.5 M NaCl/0.5 M Tris CI, pH 7.0.
  • Isofluridine (NZ-4) anti-HBV activity was verified on an animal model (Example 10). As shown in Figures 1, 2 and 3, isothiazolidine (NZ-4) induces a full-length core protein and a C-terminal missing core protein packaged into abnormal core particles. A further study showed that isothiazolidine (NZ-4) replaced pgRNA to initiate packaging and induced the formation of an empty viral nucleocapsid without HBV viral nucleic acid (Fig.

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Abstract

本发明提供了一种抗HBV的化合物的筛选方法，该方法是以HBV核心蛋白C末端氨基酸序列为药物作用靶点来筛选抗HBV的化合物，所述HBV核心蛋白C末端氨基酸序列至少包含STLPETTVVRRRDRGR。本发明提供的方法可以用于筛选和发现诱导HBV核心颗粒异常包装的抗HBV复制活性化合物，还可以用于检测以其它方式获得的针对这些靶位点的用于治疗乙型病毒性肝炎的新型抗病毒药物的活性。

Description

抗 HBV的化合物的筛选方法 技术领域 本发明属于分子生物学与生物医药技术领域， 具体而言， 本发明涉及以 乙型肝炎病毒（HBV)核心蛋白 C末端氨基酸序列为药物作用靶位点的药物 筛选方法。 背景技术 慢性乙型病毒性肝炎感染是严重危害人类健康的全球性感染疾病。 在中 国， 乙型肝炎病毒引起的肝脏疾病位列全国十大死因之一， 严重影响了人们 正常的工作和生活。 目前， 上市的小分子抗乙型肝炎病毒药物主要为核苷类 药物， 如拉米夫定、 恩替卡韦等， 尽管这些药物在抑制病毒复制上都卓有成 效， 但由于现有核苷类抗乙型肝炎病毒药物的作用靶点单一 （HBV DNA聚 合酶）， 长期使用存在的耐药突变等问题。 因此， 寻找不同于核苷类抗 HBV 药物作用机制的新靶点， 研发治疗乙型病毒性肝炎新型药物， 尤其是非核苷 类小分子抗 HBV药物， 是重要和紧迫的任务。 已报道的非核苷类小分子抗 HBV 活性化合物有： 异芳香二氢嘧啶 （HAP) 系列化合物， 代表性化合物 BAY38-7690, 它同核心蛋白的 113-143位氨基酸残基作用而干扰核心蛋白的 组装过程， 导致核心蛋白单体在细胞内过度累积而被降解； 疏水性荧光探针 bis-ANS 能与核心蛋白结合， 使之形成非壳体化的大分子多聚体， 误导核心 蛋白的正常装配过程，造成病毒基因组复制缺陷；苯丙烯酰胺类化合物 AT-61 和 AT-130能够促进核心蛋白的组装速率， 使得 pgRNA来不及被包装， 从而 使得 HBV-DNA复制水平降低； 塞菊芋黄素同系化合物 8-1能特异性地干扰 宿主细胞的核转录因子与 HBV启动子的相互作用，下调病毒 RNA的转录水 平。
HBV核心蛋白 C末端共计 34个氨基酸， 为鱼精蛋白样结构， 其中有 16 个精氨酸， 形成有 3个 SPRRR序列与 4个精氨酸聚集区， 为高碱性末端， 可结合前基因组 RNA与聚合酶复合物（pgRNA-Pol)启动核壳体包装。 目前 尚未见有报道将核心蛋白 C末端氨基酸作为药物作用靶点进行抗 HBV药物 的筛选。
重组 DNA技术是分子生物学中常用的一种生物技术， 是指将一种生物 体 （供体） 的基因与载体在体外进行拼接重组， 然后转入另一种生物体 （受 体） 内， 使之按照人们的意愿稳定遗传并表达出新产物或新性状的 DNA体 外操作程序， 也称为分子克隆技术。 本发明通过重组 DNA技术构建多种真 核表达质粒来表达 C末端缺失、截短或突变的核心蛋白， 确定核心蛋白 C末 端 4 个带正电荷的精氨酸富集区域 （RRRDRGR、 SPRRR、 SPRRRR 和 SPRRRR)为关键靶点， 以此筛选的化合物能有效的诱导形成空载病毒颗粒， 从而抑制病毒复制。 发明内容 针对现有技术中的不足， 本发明人进行了广泛深入的研究， 并最终完成 了本发明。
本发明的目的是提供一种抗 HBV的化合物的筛选方法。
根据本发明的目的， 本发明提供了一种抗 HBV 的化合物的筛选方法， 其中， 该方法以 HBV核心蛋白 C末端氨基酸序列为药物作用靶点来筛选抗 HBV 的化合物， 所述 HBV 核心蛋白 C 末端氨基酸序列至少包含 STLPETTVVRRRDRGR。
本发明中， 优选地， 所述 HBV核心蛋白 C末端氨基酸序列可为：
STLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC
STLPETTVVRRRDRGRSPRRRTPSPRRRRSQ
STLPETTVVRRRDRGRSPRRRTP或
STLPETTVVRRRDRGR。
具体地， 所述方法包括以下歩骤：
(a) 通过 PCR的方法构建含有 HBV核心蛋白 C末端氨基酸序列的真 核表达质粒， 其中， 所述 HBV 核心蛋白 C 末端氨基酸序列至少包含 STLPETTVVRRRDRGR。
(b) 用歩骤 （a) 中所得质粒瞬时转染肝癌细胞 （Huh7 细胞， HepG2 细胞等细胞系， 优选为 Huh7细胞） 4〜6小时后给予筛选化合物处理 48〜96 小时。 (c)药物处理后收集细胞， 以非变性琼脂糖凝胶电泳来筛选可诱导异常 空载 HBV核心颗粒包装的化合物。
本发明中， 优选地， 所述歩骤（b) 中筛选化合物处理为： 将转染的肝癌 细胞或与筛选化合物共同培养，所述化合物的浓度可以根据需要而进行设定。
所述歩骤（c)具体为： 经筛选化合物处理后收集细胞， 取细胞裂解液上 样于 1.8%琼脂糖凝胶孔中， 4°C电泳 2〜3小时， 优选为 2.5小时。 电泳结束 后， 虹吸法转膜过夜。 所得样品可转移至硝酸纤维素膜上， 采用与蛋白质印 迹法（Western blot)相似的方法， 用抗 HBc抗体（Abeam)检测由筛选化合 物诱导产生的异常核心颗粒。 所述异常核心颗粒是指不含 HBV病毒遗传物 质（病毒核酸）的空载 HBV核心颗粒，可阻碍 HBV病毒的进一歩感染复制。
本发明提供的筛选化合物的方法可用于筛选和发现诱导 HBV核心颗粒 的异常包装的抗 HBV复制活性化合物， 以期发现化合物作用于 HBV核心蛋 白 C末端靶序列， 诱导核心蛋白组装成为不含 HBV病毒遗传物质 （病毒核 酸） 的空载 HBV核心颗粒， 从而阻碍 HBV病毒的进一歩感染复制。 而且， 本发明提供的化合物的筛选方法还可以用于检测以其它方式获得的针对这些 靶位点的用于治疗乙型病毒性肝炎的新型抗病毒药物的活性。 相对于已有的 技术，本发明提供的筛选方法的特异性针对性更强，并且（例如与 HepG2.2.15 细胞系相比） 更有利于抗病毒药物靶点的确定。 附图说明 图 1是根据本发明的实施例 1中构建的质粒转染 Huh7细胞（实施例 6)， 而得到的核心颗粒凝胶电泳图。
图 2是根据本发明的实施例 2中构建的质粒转染 Huh7细胞（实施例 6)， 而得到的核心颗粒凝胶电泳图。
图 3是根据本发明的实施例 3中构建的质粒转染 Huh7细胞（实施例 6)， 而得到的核心颗粒凝胶电泳图。
图 4是根据本发明的实施例 4和 5中构建的质粒转染 Huh7细胞 （实施 例 6) 而得到的核心颗粒凝胶电泳图。
图 5是根据本发明的实施例 7得到的核心颗粒凝胶电泳图。
图 6是根据本发明的实施例 8得到的 HBV DNA凝胶电泳图。
图 7是根据本发明的实施例 9得到的实时荧光定量 PCR 结果图。 图 8是根据本发明的实施例 10得到的实时荧光定量 PCR 结果图。
图 9是根据本发明的实施例 1的 PCR反应程序的图。
图 10是根据本发明的实施例 2的 PCR反应程序的图。
图 11是根据本发明的实施例 9的 PCR反应程序的图。 具体实施方式 以下实施例将有助于本领域的普通技术人员进一歩理解本发明， 但不以 任何形式限制本发明。 实施例 1 : 构建针对靶序列 HBc-1 的全长 HBV核心蛋白真核表达质粒 pHBcl85
以表达 1.3倍全长 HBV基因组 （adw 亚型） pHBV1.3为模板， 克隆编 码全长核心蛋白的 DNA片段和 pcDNA3.1质粒（购自 Invitrogen )重组， 转 化到 受体菌 DH5a (购自天根生化科技有限公司） 中， 经筛选和鉴定， 得到了全长核心蛋白的真核表达质粒 pHBcl85。 扩增引物如下：
SP/HBc 1 Pstl ( SEQ ID NO: 6)
5 ' -TTTTCTGC AGATGGAC ATTC
ASP/HBc 185 Pst 1 ( SEQ ID NO: 7)
5'-TTT
GACG-3O
PCR 反应程序如图 9所; 转化条件如下:
4。C， 30min
42°C t 90s
4°C, 5min 质粒 pHBcl85可在真核细胞中表达全长的 HBV核心蛋白， 其 C末端包 括四个精氨酸富集区，其中三个为 SPRRR重复序列，序列为 HBc-1 (SEQ ID NO: 1)：
STLPETTVVRRRDRGRSPR RTPSPRRRRSQSPRRRRSQSRESQC。 实施例 2: 构建针对靶序列 HBc-2的 C末端截短 HBV核心蛋白真核表 达质粒 pHBc l
以实施例 1制备的 pHBcl85为模板，通过 PCR产生载体片段 pHBCTD, 扩增引物如下：
SP/pHBcl85/1502-1401 (SEQ ID NO: 8)
5'-TCGGGAATCTCAATGTTAGCTGC-3 '，
ASP/pHBcl85/1502-1401 (SEQ ID NO: 9)
5 ' -TAGTTTCCGGAAGTGTTGATAAGATAGGGGCATTTG-3 '，
合成编码 HBV核心蛋白 C末端 141-171位氨基酸的 DNA插入片段，序 （SEQIDNO: 10)：
Figure imgf000006_0001
CGCAGACGAAGGTCTCAA -3Ό
利用同源重组技术将合成片段插入上述通过 PCR 产生的载体片段 pHBCTD 中， 转化到 E.coli受体菌 （同实施例 1) 中， 经筛选和鉴定， 得到 了 C末端截短核心蛋白真核表达质粒 pHBcl71。
PCR 反应程序如图 10所示。 同源重组反应条件如下:
37。Ct 15min
50°Gt ISniiii
转化条件同实施例 1。 质粒 pHBc l可在真核细胞中表达 C末端部分缺失的核心蛋白， 其 C 末端包括两个精氨酸富集区，缺失两个 SPRRR重复序列，序列为 HBc-2( SEQ ID NO: 2)：
STLPETTVVRRRDRGRSPRRRTPSPRRRRSQo
实施例 3 : 构建针对靶序列 HBc-3的 C末端截短 HBV核心蛋白真核表 达质粒 pHBcl63
合成编码 HBV核心蛋白 C末端 141-163位氨基酸的 DNA插入片段，序 （SEQ ID NO: 11 )：
Figure imgf000007_0001
利用同源重组技术将合成片段插入上述通过 PCR 产生的载体片段 pHBCTD 中， 转化到 E.coli受体菌 （同实施例 1 ) 中， 经筛选和鉴定， 得到 了 C末端截短核心蛋白真核表达质粒 pHBcl63。
PCR 反应程序同实施例 2。
同源重组反应条件同实施例 2。
转化条件同实施例 1。
质粒 pHBcl63可在真核细胞中表达 C末端部分缺失的核心蛋白， 其 C 末端包括两个精氨酸富集区，缺失两个 SPRRR重复序列，序列为 HBc-3( SEQ ID NO: 3 )：
STLPETTVVRRRDRGRSPRRRTP。 实施例 4: 构建针对靶序列 HBc-4的 C末端截短 HBV核心蛋白真核表 达质粒 pHBcl56
合成编码 HBV核心蛋白 C末端 141-156位氨基酸的 DNA插入片段，序 列如下 （SEQ ID NO: 12)：
GGCAATCTCGGGAATCTC AATG-3 ' 利用同源重组技术将合成片段插入实施例 2中通过 PCR产生的载体片段 pHBCTD 中， 转化到 E.coli受体菌 （同实施例 1 ) 中， 经筛选和鉴定， 得到 了 C末端截短核心蛋白真核表达质粒 pHBcl56。
PCR 反应程序同实施例 2。
同源重组反应条件同实施例 2。
转化条件同实施例 1。
质粒 pHBcl56可在真核细胞中表达 C末端部分缺失的核心蛋白， 其 C 末端只有一个精氨酸富集区，缺失三个 SPRRR重复序列，序列为 HBc-4( SEQ ID NO: 4)：
STLPETTVVRRRDRGR。 实施例 5: C末端缺失 HBV核心蛋白真核表达质粒 pHBcl56M的构建 合成编码 HBV核心蛋白 C末端 141-156位氨基酸的 DNA插入片段，并 将 150、 151、 152、 154和 156位精氨酸由丙氨酸替代，序列如下（SEQ ID NO:
13 )：
CACAATCTCGGGAATCTCAATG-3'。
利用同源重组技术将合成片段插入载体 pHBCTD中，转化到 E.coli受体 菌 （同实施例 1 ) 中， 经筛选和鉴定， 得到了 C末端截短 HBV核心蛋白真 核表达质粒 pHBcl56M。
PCR 反应程序同实施例 2。
同源重组反应条件同实施例 2。
转化条件同实施例 1。
质粒 pHBcl56M可在真核细胞中表达 C末端突变的核心蛋白， 其 C末 端没有精氨酸富集区， 序列为：
STLPETTVVAAADAGA ( SEQ ID NO: 5 )。 实施例 6: HBV核心蛋白真核表达质粒瞬时转染 Huh 7细胞
Huh 7细胞 （人肝癌细胞系， 中科院武汉病毒所馈赠， 本室保存） 培养 于含 10%FBS的 DMEM培养液中， 5% C0237°C恒温培养。在进行质粒转染 实验时， Huh 7细胞以 5xl05个细胞 /孔的密度接种于 6孔板中贴壁过夜。 次 日， 根据 Lipofectamine 2000 Transfection Reagent (Invitrogen)转染试齐 (J说明书 采用上述实施例 1、 2、 3、 4和 5制备的相应的真核表达质粒进行质粒转染实 验。 转染六小时后， 更换含相应浓度的待测化合物 （本发明选用根据现有技 术由本所合成的抗乙肝病毒药物异噻氟定 （NZ-4)作为待测化合物 （其终浓 度为 20微摩尔、 10微摩尔或 5微摩尔） 的 DMEM培养液进行培养。 培养 48小时后， 细胞用于相应检测。 实施例 7: 非变性琼脂糖凝胶电泳筛选抗 HBV的活性化合物
分别将 HepG2.2.15细胞（中科院武汉病毒所馈赠， 本室保存）或实施例 6中制备的质粒瞬时转染的 Huh 7细胞与不同浓度的待测化合物 （即终浓度 为 20微摩尔、 10微摩尔或 5微摩尔的异噻氟定） 共同培养后， 收集细胞， 细胞裂解液经离心去除细胞核和细胞碎片后， 上样于 1.8%琼脂糖凝胶孔中， 4°C电泳 2.5小时。 电泳结束后， 虹吸法转膜过夜。 样品可转移至硝酸纤维素 膜上， 采用与 Western blot相似的方法， 用抗 HBc抗体 （Abeam) 检测核心 颗粒。 也可在电泳结束后用变性液（1.5 M NaCl/0.5 M NaOH)进行凝胶原位 变性 45 min， 中和液 （ 1.5 M NaCl/0.5 M Tris CI, pH 7.0) 中和 45 min， 再转 移至 Hybond-N+ 尼龙膜上，后续方法与 DNA印迹法（Southern blot)和 RNA 印迹法 （Northern blot) 相似， 用 HBV特异性探针 （达安基因公司） 检测核 壳体内的核酸水平。 其中， HepG2.2.15细胞是由人肝癌细胞系 HepG2细胞 衍生的， 能够稳定转染 HBV基因、 稳定进行 HBV基因组复制、 稳定表达 HBV, 可对通过本方法筛选出的化合物的抗 HBV活性加以验证， 同时还可 用来检测化合物对 HBV DNA的影响。
上述实施例 1中构建的质粒 pHBcl85转染 Huh7细胞进行化合物筛选而 得到的核心颗粒凝胶图电泳如图 1所示， 该实验结果表明通过筛选得到的化 合物 NZ-4可结合于核心蛋白 C末端精氨酸富集区，诱导异常核心颗粒形成， 这一作用不依赖于 pgRNA的参与。
上述实施例 2中构建的质粒 pHBc l转染 Huh7细胞进行化合物筛选而 得到的核心颗粒凝胶电泳图如图 2所示， 该实验结果表明 NZ-4可诱导 C末 端截短的核心蛋白形成异常核心颗粒。 上述实施例 3中构建的质粒 pHBcl63转染 Huh7细胞进行化合物筛选而 得到的核心颗粒凝胶电泳图如图 3所示， 该验结果表明 NZ-4可诱导 C末端 截短的核心蛋白形成异常核心颗粒。
上述实施例 4和 5中构建的质粒 pHBcl56和 pHBcl56M转染 Huh7细胞 进行化合物筛选而得到的核心颗粒凝胶电泳图如图 4所示， 该实验结果表明 NZ-4对 C末端精氨酸突变为丙氨酸的核心蛋白无效， 说明该区域带正电荷 的精氨酸为药物筛选的关键靶点。
如图 5显示， HepG2.2.15细胞上 NZ-4 诱导产生的异常核心颗粒为空载 核心颗粒， 不含病毒核酸， 核壳体内 DNA水平显著降低。 其中， GAPDH为 甘油醛 -3-磷酸脱氢酶，其基因为看家基因，几乎在所有细胞中都高水平表达。 在同种细胞中 GAPDH蛋白表达量一般是恒定的， 因此在该实施例中被作为 实验操作的标准化的内参。 实施例 8: DNA印迹法 （Southern blotting) 确定化合物抗 HBV活性 HepG2.2.15细胞裂解液经离心除去细胞核和细胞碎片， 核酸酶剪切后加 入蛋白酶 K消化， 经酚氯仿抽提， 异丙醇沉淀得到病毒 DNA。将 DNA上样 于 0.8%琼脂糖凝胶孔中， 70V 电泳 1 小时。 电泳结束后用变性液 （1.5 M NaCl/0.5 M NaOH) 进行凝胶原位变性 45 min, 中和液 （1.5 M NaCl/0.5 M Tris CI, H 7.0) 中和 45 min， 转移至 Hybond-N+ 尼龙膜上， 紫外交联 （子 外交联仪， 宁波新芝生物科技股份有限公司） 后用地高辛标记的 HBV特异 性抗体 （Roche) 杂交。 图 6示出了得到的 HBV DNA凝胶电泳图， 该实验 结果表明在 HepG2.2.15细胞上 NZ-4抑制病毒的三种复制中间体， 降低细胞 内 HBV-DNA水平。 实施例 9: 实时荧光定量 PCR (Real-time RCR) 检测细胞培养上清中病 毒粒子数量
HepG2.2.15与不同浓度的待测化合物共同培养 8天后， 收集细胞培养上 清按照 Dneasy Tissue Kit试剂盒说明书进行（Qiagen)提取病毒 DNA, 进行 荧光定量 PCR检测。
PCR 反应程序如图 11所示。 图 7示出了得到的实时荧光定量 PCR 结果图，实验结果表明 NZ-4降低 HepG2.2.15细胞培养上清中病毒粒子的水平。
实施例 10: HBV转基因小鼠模型 （Balb/c背景）
6周龄的 HBV转基因小鼠 （购自广州解放军第 458医院， 肝病研究所） 分为四组： 模型组 （制剂辅料对照治疗组）； 100mg/kg, 50mg/kg和 25mg/kg 异噻氟定 （NZ-4) 治疗组， 口服 （灌胃） 给药。 每组 6-7只小鼠， 连续给药 治疗 28天， 分别在给药前 （0d)、 给药后第 7天 （7d)、 第 14天 （14d)、 第 21天 （21d) 和第 28天 （28d) 进行眼底静脉丛取血， 收集小鼠血清， 提取 其中的 HBV DNA进行荧光定量 PCR检测 （同实施例 9)。
图 8示出了得到的实时荧光定量 PCR 结果图，表明 NZ-4可显著降低 HBV 转基因小鼠血清中的 HBV DNA水平
实验结果：
一方面， 本发明用质粒 pHBcl85瞬时转染 Huh7细胞的方法表达全长核 心蛋白， 以非变性琼脂糖凝胶电泳来筛选可诱导异常空载核心颗粒包装的化 合物， 确定 HBV核心蛋白 C末端 45个氨基酸（aal41-aal85， adw亚型） 为 抗病毒药物筛选的靶序列， 其序列为 SEQ ID NO: l :
Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly 1 5 10 15
Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser
20 25 30
Gin Ser Pro Arg Arg Arg Arg Ser Gin Ser Arg Glu Ser Gin Cys
35 40 45 另一方面，本发明用质粒 pHBc l瞬时转染 Huh7细胞的方法表达 C末 端截短核心蛋白， 以非变性琼脂糖凝胶电泳来筛选可诱导异常核心颗粒包装 的化合物， 确定核心蛋白 C末端截短序列（aal41-aal71 )为抗病毒药物筛选 的靶序列， 其序列为 SEQ ID NO: 2：
Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser
20 25 30
又一方面，本发明用质粒 pHBcl63瞬时转染 Huh7细胞的方法表达 C末 端截短核心蛋白， 以非变性琼脂糖凝胶电泳来筛选可诱导异常核心颗粒包装 的化合物， 确定核心蛋白 C末端截短序列（aal41-aal63 )为抗病毒药物筛选 的靶序列， 其序列为 SEQ ID NO: 3 :
Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly 1 5 10 15
Arg Ser Pro Arg Arg Arg Thr Pro
20 再一方面，本发明用质粒 pHBcl56瞬时转染 Huh7细胞的方法表达 C末 端截短核心蛋白， 以非变性琼脂糖凝胶电泳来筛选可诱导异常核心颗粒包装 的化合物， 确定核心蛋白 C末端截短序列（aal41-aal56)为抗病毒药物筛选 的靶序列， 其序列为 SEQ ID NO: 4：
Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg
1 5 10 另外， 本发明核心蛋白 C末端一个精氨酸富集区域（aal50-aa156)为抗 病毒药物筛选的靶序列， 但如果将其中的 5个精氨酸突变为丙氨酸后得到的 C末端突变核心蛋白不再具有筛选抗 HBV化合物的活性 （如实施例 7中对 质粒 pHBcl56和 pHBcl56M的描述）， 所述的精氨酸富集区域的序列为 Arg Arg Arg Asp Arg Gly Arg
1 5 表 1为根据上述实施例确定的 HBV (adw亚型)的核心蛋白 C末端靶序列， 从上至下依次对应于实施例 1、 2、 3和 4。 表 1
序列 靶序列 靶序列长度 位置 编号 (aa) (aa)
HBc-1 STLPETTWRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 45 141-185
STLPETTWRRRDRGRSPRRRTPSPRRRRSQ
HBc-3 STLPETTWRRRDRGRSPRRRTP 23 141-163
HBc-4 STLPETTWRRRDRGR 16 141-156
实施例 11 : 非核苷类小分子化合物异噻氟定 (NZ-4)的抗 HBV活性的检 利用本发明所述的筛选方法发现和证实了非核苷类小分子化合物异噻氟 定 (NZ-4)具有显著的抗 HBV活性和新颖的抗病毒作用机制。主要检测歩骤如 下：
( 1 ) HepG2.2.15细胞贴壁 24小时后分别给予终浓度为 20微摩尔、 10 微摩尔和 5微摩尔的异噻氟定 (NZ-4)处理 8天。
(2 ) 收集细胞， 以非变性琼脂糖凝胶电泳的方法检测异噻氟定 (NZ-4) 诱导产生异常空载核心颗粒 （实施例 7)。
具体方法是取细胞裂解液上样于 1.8%琼脂糖凝胶孔中， 4°C电泳 2.5小 时。 电泳结束后， 虹吸法转膜过夜。 样品可转移至硝酸纤维素膜上， 采用与 Western blot相似的方法， 用抗 HBc抗体 （Abeam) 检测由筛选化合物诱导 产生的异常核心颗粒。 同时， 制备相同样品进行凝胶电泳， 电泳结束后用变 性液 （ 1.5 M NaCl/0.5 M NaOH) 进行凝胶原位变性 45 min, 中和液 （ 1.5 M NaCl/0.5 M Tris CI, pH 7.0) 中和 45 min， 再转移至 Hybond-N+ 尼龙膜上， 后续方法与 Southern blot和 Northern blot相似，用 HBV特异性探针检测核壳 体内的核酸水平。 (3 ) 用上述实施例 1〜5制备的质粒 pHBcl85、 pHBcl71、 pHBcl63、 pHBcl56和 pHBcl56M分别瞬时转染 Huh7细胞六小时后分别给予终浓度为 10微摩尔和 5微摩尔的异噻氟定 (NZ-4)处理 48小时 （实施例 6)。
(4) 收集细胞， 以非变性琼脂糖凝胶电泳的方法确定异噻氟定 (NZ-4) 诱导异常空载核心颗粒产生的主要作用靶序列 （实施例 7)。
( 5 )通过 Southern blot (实施例 8)和实时荧光定量 PCR (实施例 9) 对异噻氟定 (NZ-4)抗 HBV活性加以验证。
(6) 在动物模型上验证异噻氟定 (NZ-4)抗 HBV活性 （实施例 10)。 如图 1、 2和 3所示，异噻氟定 (NZ-4)可诱导全长的核心蛋白和 C末端缺 失的核心蛋白包装成异常核心颗粒。 进一歩研究表明异噻氟定 (NZ-4)取代 pgRNA启动包装， 诱导形成不含 HBV病毒核酸的空载病毒核壳体 （图 5 ) ; HBV核心蛋白 C末端的五个精氨酸序列是异噻氟定 (NZ-4)的主要作用靶点， 将其突变成不带电荷的丙氨酸后，异噻氟定 (NZ-4)不在发挥抗 HBV作用（图 4)。 其可能的作用机制为异噻氟定 (NZ-4)通过与核心蛋白 C末端精氨酸富集 区相互作用， 结合于该区域而抑制 pgRNA包装， 产生不含病毒核酸的空载 病毒颗粒， 而失去 pgRNA模板的核壳体无法完成逆转录过程， 从而降低了 细胞内 HBV-DNA的复制水平（图 6);空载病毒颗粒被证明虽能分泌出细胞， 但由于缺乏病毒遗传物质 （病毒核酸）， 细胞培养上清中的 HBV DNA水平 也显著降低 （图 7)。 同时， HBV空载病毒颗粒不能也无法进行新一轮的感 染和复制。 在体内抗 HBV病毒复制的药效学研究中， NZ-4 在 HBV转基因 小鼠模型上表现出明显的抗病毒复制活性 （图 8)。

Claims

权 利 要 求
1、 一种抗 HBV的化合物的筛选方法， 其中， 该方法以 HBV核心蛋白 C末端氨基酸序列为药物作用靶点来筛选抗 HBV的化合物， 所述 HBV核心 蛋白 C末端氨基酸序列至少包含 STLPETTVVRRRDRGR。
2、 根据权利要求 1所述的方法， 其中， 所述 HBV核心蛋白 C末端氨基 酸序列为：
STLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC STLPETTVVRRRDRGRSPRRRTPSPRRRRSQ STLPETTVVRRRDRGRSPRRRTP或
STLPETTVVRRRDRGR。
3、 根据权利要求 1所述的方法， 其中， 所述方法包括以下歩骤：
(a) 通过 PCR的方法构建含有所述 HBV核心蛋白 C末端氨基酸序列 的真核表达质粒；
(b)用歩骤（a)中所得质粒瞬时转染肝癌细胞 4〜6小时后给予筛选化 合物处理 48〜96小时；
(c)药物处理后收集细胞， 以非变性琼脂糖凝胶电泳来筛选可诱导异常 空载 HBV核心颗粒包装的化合物。
4、 根据权利要求 3所述的方法， 其中， 所述肝癌细胞包括 Huh7细胞或 HepG2细胞。
5、 根据权利要求 4所述的方法， 其中， 所述肝癌细胞为 Huh7细胞。
6、 根据权利要求 3所述的方法， 其中， 所述歩骤 （b) 中筛选化合物处 理为： 将转染的肝癌细胞与筛选化合物共同培养。
7、 根据权利要求 3所述的方法， 其中， 所述歩骤 （c) 具体为： 经筛选 化合物处理后收集细胞， 取细胞裂解液上样于 1.8%琼脂糖凝胶孔中， 4°C电 泳 2〜3小时； 电泳结束后， 虹吸法转膜过夜； 将所得样品转移至硝酸纤维素 膜上， 用抗 HBc抗体检测由筛选化合物诱导产生的异常核心颗粒。
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ASIF-ULLAH M. ET AL.: "Identification of compounds that inhibit the interaction between core and surface protein of hepatitis B virus.", ANTIVIRAL RESEARCH., vol. 70, 2006, pages 85 - 90, XP025031476, DOI: doi:10.1016/j.antiviral.2006.01.003 *
HU, XUEJUN.: "Design, synthesis and screening of novel anti-HBV agents targeting at capsid assembly.", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE: MEDICINE AND HEALTH SCIENCES., 15 July 2010 (2010-07-15), pages E079 - 2 *
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