Drug Evaluation Model Of HBV Molecular Pharmacology And The Strategies To Reduce Nucleosides/Nucleotides Resistance

CccDNA is the molecular basis of the persistent HBV infection. It plays a critical role in viral rebound after treatment withdrawal as well as in drug resistance. Presently, the main mission for HBV treatment is to develop novel antivirus-strategies to prevent the formation of cccDNA or decrease the cccDNA pool in hepatocytes.The intracellular cycle of cccDNA proliferation involves in all of the steps in HBV replication. There are several questions remained uncovered. Which step is the key to suppress cccDNA synthesis? Some of the anti-HBV drugs have a low chance to induce NAs drug-resistance. What is their effect and mechanism on cccDNA inhibition? Investigation of these issues will help us to elucidate the clinical drug-resistance, discover high efficient drugs to inhibit cccDNA reduplication, and develop new methods for the combination therapy. For this purpose, small-molecule anti-HBV compound drugs were utilized as probes in this work.OMTR, PFA,3TC, ADV and ETV are clinically effective against HBV. They are small molecular weight compounds with different structures, mode of action, targets or binding target sites. OMTR is a selective inhibitor for host HSC70 mRNA stability. PFA had a high affinity to the pyrophosphate binding site in the viral polymerase.3TC, ADV and ETV target the viral polymerase but with different binding modes. Based on the distinctive mechanism of these compounds in their anti-HBV action, one of our major goals is to study their mechanisms in inhibiting cccDNA replication. This might help us to reduce HBV drug-resistance through inhibition of cccDNA proliferation.A series of new constructed qPCR were exploited to detect the major replication steps in the asymmetric replication process of HBV, which including minus-strand replication, plus-strand replication and cccDNA synthesis. The cccDNA proliferation is the primary molecular event of HBV replication in HepG2.2.15 cells after cell dividing. This cell line is an efficient model to study the mechanisms of anti-HBV drugs due to the lack of negative feedback regulation of envelope protein for cccDNA proliferation in cccDNA proliferation.As a result,'drug evaluation model of HBV molecular pharmacology'is proposed. Based on the quantitative detection of the key proliferative steps of cccDNA by the series of qPCR, the model studies drug mechanisms of action in HBV cccDNA proliferation. In combination with pharmacodynamic and partial correlation analysis, the model could be used to evaluate primary or secondary effect of minus-strand and plus-strand replication in the cccDNA proliferation. In short, this model is a multi-level evaluation system, applying the HBV feature mechanism of asymmetric replication into the study of HBV pharmacodynamics. The goal is to understand the major molecular pharmacodynamics of drugs in the whole cycle of HBV replication.3TC, ADV and ETV effectively inhibit cccDNA synthesis in HepG2.2.15 cells. Because HBsAg and cccDNA are biologically relevant, the extracellular HBsAg level was also measured quantitatively in the present research. The results suggested that HBsAg level quantitatively and qualitatively reflected the amount of cccDNA in the antiviral treatment, and confirmed the suppression of cccDNA after the antiviral treatment.The dose-dependent inhibition curves of HBV minus-strand replication, plus-strand replication and cccDNA replication were analyzed, including the pharmodynamic and partial correlation analysis. Collectively, our data demonstrated that NAs suppressed HBV cccDNA proliferation through different mechanisms.3TC, ETV or ADV inhibits cccDNA proliferation at the stage of minus-strand replication, plus-strand replication or plus-strand complementary stage respectively. The mechanism of ETV and ADV were confirmed by further experiments.HSC70 is a replicase-activating factor in HBV reverse transcription. In our previous research, natural compound oxymatrine (OMTR) was found to be a selective inhibitor of HSC70 expression and the anti-HBV effect of OMTR was mediated through destabilizing HSC70 mRNA. Furthermore, the coiled coil region on HSC70 was revealed to be the critical motif for HBV replication. Distinct from NAs, OMTR equally decreased the replication of minus strand, plus strand and cccDNA. It was considered that the anti-cccDNA effect of OMTR appeared to be mediated through inhibiting minus-strand replication.Anti-HBV activity of PFA was relative weak. The efficacious feature of PFA was different from OMTR. Its inhibiting efficacy follows the order of minus-strand replication< plus-strand replication< cccDNA synthesis. It was suggested that PFA might suppress cccDNA synthesis at the stage of the plus-strand complementary phase.The effect of combination therapies with 3TC, ADV and ETV was studied in this project as well. The combination of 3TC with ADV was the only regimen that suppressed intracellular HBV and the proliferation of cccDNA synergistically.In conclusion,'drug evaluation model of HBV molecular pharmacology'has significant value in anti-HBV research. It could be applied to study molecular pharmaco-dynamics of anti-HBV drugs, and help us to find the way to fight against HBV drug resistance.