Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects over 350 million people worldwide and causes 600,000 deaths annually. Current therapies for HBV infection use interferon 1 or one of five nucleoside analogs. Neither form of treatment cures HBV infections and both have major limitations. Furthermore, the nucleoside analogs all target the DNA polymerase active site on the viral reverse transcriptase, and hence cross-resistance between some of the drugs is common. Therefore, novel antivirals that act on targets other than the DNA polymerase active site are urgently needed. We recently identified two motifs called "T3" and "RT1" on the Duck Hepatitis B virus (DHBV) reverse transcriptase that together form the site where the enzyme first binds to the RNA form of the viral genome. Disrupting RNA binding at the T3:RT1 site blocks reverse transcription. Neither T3 nor RT1 are in the DNA polymerase active site, and consequently inhibitors of the T3:RT1 site would function by a novel mechanism and hence would be perfect candidates for combination therapy with the nucleoside analog drugs. The T3 and RT1 motifs were identified through their high homology between DHBV and HBV, and mutating the HBV T3 motif ablates DNA synthesis, but little work has been done with the HBV enzyme itself. Therefore, in this Phase I STTR project we will extend our analysis of RNA binding by the DHBV reverse transcriptase to the HBV enzyme. The goal of this application is to identify the form of the HBV enzyme best suited for subsequent high through-put screening for novel anti-T3:RT1 drugs. Aim 1. Evaluate the role of the HBV T3 and RT1 motifs in RNA binding. In vitro RNA binding assays with recombinant HBV reverse transcriptase will be done to determine how well data obtained with the DHBV enzyme reflects RNA binding at the HBV T3:RT1 site. These results will be confirmed in vivo by evaluating the effects of mutations to the HBV T3 and RT1 motifs on encapsulation of the HBV genomic RNA. Aim 2. Determine the optimal configuration for an HBV reverse transcriptase RNA binding assay amenable to drug screening. We will adapt our current filter-binding assay that measures RNA binding by the DHBV reverse transcriptase to the medically-relevant HBV enzyme. In Phase II of this STTR project we will fully develop an HBV reverse transcriptase RNA binding assay into a 384-well format employing fluorescent detection of the RNA and conduct a pilot screen of negatively-charged non-nucleoside compounds for inhibitors of the RNA binding reaction. We will then seek a major pharmaceutical partner for full-scale high-throughput screening.
Public Health Relevance: Hepatitis B virus replicates by reverse transcription, kills 600,000 people each year, and current therapies for the infection are inadequate. Using an animal model for HBV, we recently identified the site on the viral reverse transcriptase where the enzyme first binds to its viral RNA template and demonstrated that disrupting this site blocks viral replication. Here, we will extend these results to the medically-relevant HBV enzyme, with the goal of identifying the format of an assay best suited for subsequent high through-put screening for novel anti-HBV drugs.
Public Health Relevance Statement: Hepatitis B virus replicates by reverse transcription, kills 600,000 people each year, and current therapies for the infection are inadequate. Using an animal model for HBV, we recently identified the site on the viral reverse transcriptase where the enzyme first binds to its viral RNA template and demonstrated that disrupting this site blocks viral replication. Here, we will extend these results to the medically-relevant HBV enzyme, with the goal of identifying the format of an assay best suited for subsequent high through-put screening for novel anti-HBV drugs.
Project Terms: AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Active Sites; Adverse effects; Antiretroviral Therapy, Highly Active; Antiviral Agents; Antiviral Drugs; Antivirals; Assay; B virus infection; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Cessation of life; Charge; Chlorohemin; Cirrhosis; Combined Modality Therapy; DHBV; DNA Polymerases; DNA Replication; DNA Synthesis; DNA Viruses; DNA biosynthesis; DNA-Dependent DNA Polymerases; DNA-Directed DNA Polymerase; Data; Death; Deoxynucleoside-triphosphate[{..}]DNA deoxynucleotidyltransferase (DNA-directed); Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Detection; Development; Drug Evaluation, Preclinical; Drug Screening; Drug resistance; Drugs; Duck Hepatitis B Virus; EC 2.7.7.49; EC 2.7.7.7; Enzymes; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Ferrate(2-), chloro(7,12-diethenyl-3,8,13,17-tetramethyl-21H,23H-porphine-2,18-dipropanoato(4-)-N21,N22,N23,N24)-, dihydrogen, (SP-5-13)-; Ferriheme Chloride; Ferriprotoporphyrin IX Chloride; Fluorescent Probes; Gene Products, RNA; Genetic Alteration; Genetic Change; Genetic defect; Genomics; Goals; HAART; HBV; HBV Animal Model; HBV polymerase; HBV-P protein; HIV; HTLV-III; Hemin; Hepatic Cancer; Hepatitis; Hepatitis B Virus; Hepatitis B Virus, Duck; Hepatitis Virus, Homologous Serum; Herpes simiae infection; Herpesvirus B infection; High Throughput Assay; Highly Active Antiretroviral Therapy; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; IFN; In Vitro; Infection; Interferons; Killings; LAV-HTLV-III; Lymphadenopathy-Associated Virus; Malignant neoplasm of liver; Measures; Medication; Minority; Modeling; Molecular Interaction; Multimodal Therapy; Multimodal Treatment; Multimodality Treatment; Mutate; Mutation; Nucleosides; P protein, hepatitis B virus; Patients; Peptides; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Preclinical Drug Evaluation; Protohemin; Protohemin IX; RNA; RNA Binding; RNA Transcriptase; RNA, Non-Polyadenylated; RNA, Viral; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Reaction; Recombinants; Resistance; Reverse Transcriptase; Reverse Transcription; Revertase; Ribonucleic Acid; Role; STTR; Screening procedure; Simian B disease; Simian B disorder; Simian B virus infection; Site; Small Business Technology Transfer Research; Treatment Side Effects; Viral; Viral Diseases; Viral Genome; Virus; Virus Diseases; Virus-HIV; Viruses, General; Work; anti-hepatitis B; anti-retroviral therapy, highly active; combination therapy; combined modality treatment; combined treatment; drug resistant; drug/agent; genome mutation; hepatitis B virus P protein; high throughput screening; in vivo; inhibitor; inhibitor/antagonist; liver cancer; malignant liver tumor; multimodality therapy; novel; nucleoside analog; nucleotide analog; public health relevance; resistance to Drug; resistant; resistant to Drug; screening; screenings; side effect; small molecule; social role; therapy adverse effect; treatment adverse effect; viral RNA; viral infection; virus RNA; virus infection
