Hepatitis B Virus diversity and ribonuclease H inhibitor efficacy

乙型肝炎病毒多样性和核糖核酸酶 H 抑制剂功效

• 批准号: 8701533
• 负责人: JOHN E TAVIS
• 金额: $ 7.58万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701533
• 关键词: Active Sites; Affect; Antiviral Agents; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Clinical; Combined Modality Therapy; Communication; DNA; DNA Viruses; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Detection; Drug Targeting; Drug resistance; Enzymes; Evolution; Genetic Variation; Genotype; Geographic Distribution; HBV Genotype; Hepatitis B Virus; Infection; Life; Maintenance; Mutation; Patients; Pharmaceutical Preparations; Phylogenetic Analysis; Preclinical Drug Evaluation; Proteins; RNA-Directed DNA Polymerase; Recombinants; Research Project Grants; Reverse Transcription; Ribonuclease H; Testing; Trees; Variant; Viral; Viral Genome; Virus; analog; anti-hepatitis B; drug candidate; drug development; inhibitor/antagonist; killings; nucleoside analog; prevent; public health relevance; resistance mutation; screening; success; viral DNA; viral RNA; virus genetics

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >350 million people and kills up to 1.2 million patients annually. Therapy primarily employs nucleos(t)ide analogs. These drugs profoundly suppress HBV but they rarely cure patients due to incomplete inhibition of viral replication. Furthermore, control of HBV often fails due to evolution of drug resistance mutations. Pharmacologically curing HBV in more patients should be possible by suppressing HBV further with new drugs to be used in combination with the nucleos(t)ide analogs. HBV reverse transcription requires 2 viral enzymatic activities that are both located on the viral reverse transcriptase protein: the DNA polymerase that is targeted by the nucleos(t)ide analogs and the ribonuclease H (RNAseH) that destroys the viral RNA after it has been copied into DNA. Anti-HBV RNAseH drugs have not been developed because enzyme suitable for drug screening could not be made. We recently made active HBV RNAseH and identified 13 inhibitors of the RNAseH. HBV has 8 genotypes (A-H), and anti-RNAseH drugs must inhibit a wide range of HBV strains to be clinically effective. We found that RNAseH inhibitors identified against genotype D and H isolates can block replication of a genotype A isolate, so cross-genotypic inhibition is possible. However, the genotype D and H RNAseH isolates we tested are differentially sensitive to RNAseH inhibitors. We do not know whether these dissimilarities are due to genotype-specific or isolate-specific differences because have examined only 1 isolate for each genotype. Furthermore, we have not characterized recombinant RNAseH from genotypes B and C, which are the most medically-relevant genotypes. In this Small Research Grant (R03) project we will evaluate the degree to which HBV's high genetic variation affects its sensitivity to RNAseH inhibitors. Aim 1: Evaluate how differences between and within HBV's genotypes affect sensitivity to RNAseH inhibitors. We will test sensitivity of variant HBV RNAseH sequences from genotypes B, C, and D to a set of RNAseH antagonists in biochemical and viral replication assays. Aim 2: Determine how nucleoside analog resistance mutations affect sensitivity to RNAseH inhibition. We will introduce common nulceos(t)ide analog resistance mutations into HBV genotype B, C, and D isolates and evaluate how they affect sensitivity of viral replication to an RNAseH inhibitor. This project will: 1) Reveal whether HBV genotypic differences will complicate anti-RNAseH drug development; 2) Help determine whether communication between the 2 active sites on the reverse transcriptase may complicate combination therapy with nucleos(t)ide analogs and RNAseH inhibitors; and 3) Generate a set of variant HBV RNAseHs for screening drug candidates for cross-genotypic efficacy.

B型肝炎病毒（HBV）是一种嗜肝DNA病毒，通过逆转录进行复制。长期以来， 每年感染超过3.5亿人并杀死多达120万患者。治疗主要采用核苷（酸） 类似物这些药物能显著抑制HBV，但由于对HBV的不完全抑制， 病毒复制此外，由于耐药性突变的演变，HBV的控制常常失败。 通过新药进一步抑制HBV的产生，使更多的HBV患者获得药物治疗成为可能 与核苷类似物联合使用。 HBV逆转录需要两种病毒酶活性，这两种酶活性都位于病毒逆转录酶上。 转录酶蛋白：被核苷类似物和核糖核酸酶H靶向的DNA聚合酶 RNA酶（RNAseH）在病毒RNA复制成DNA后将其破坏。抗HBV RNAseH药物没有 由于不能制备适合于药物筛选的酶，因此开发了这种酶。我们最近让 HBV RNA酶H的抑制剂，并确定了13个RNA酶H的抑制剂。 HBV有8种基因型（A-H），抗RNAseH药物必须抑制广泛的HBV毒株，才能有效地抑制HBV。 临床有效。我们发现针对基因型D和H分离株鉴定的RNAseH抑制剂可以阻断 基因型A分离株的复制，因此交叉基因型抑制是可能的。然而，基因型D和H 我们测试的RNAseH分离物对RNAseH抑制剂的敏感性不同。我们不知道这些是否 差异是由于基因型特异性或分离物特异性差异，因为只检查了1 分离每个基因型。此外，我们还没有表征来自基因型B的重组RNAseH 和C，这是医学上最相关的基因型。在这个小型研究补助金（R 03）项目中，我们将 评估HBV的高度遗传变异影响其对RNAseH抑制剂敏感性的程度。 目的1：评估HBV基因型之间和内部的差异如何影响对RNAseH的敏感性 抑制剂的我们将检测来自基因型B、C和D的变异HBV RNAseH序列对一组 生物化学和病毒复制测定中的RNAseH拮抗剂。 目的2：确定核苷类似物耐药突变如何影响对RNAseH的敏感性 抑制作用我们将在HBV基因型B、C和C中引入常见的核苷（酸）类似物耐药突变， D分离物，并评估它们如何影响病毒复制对RNAseH抑制剂的敏感性。 该项目将：1）揭示HBV基因型差异是否会使抗RNAseH药物复杂化 开发; 2）帮助确定2个活动站点之间的通信是否反向 转录酶可能使与核苷（酸）类似物和RNAseH抑制剂的联合治疗复杂化;和3） 生成一组变异HBV RNAseH，用于筛选交叉基因型疗效的候选药物。