Our goal is to develop a new cell-based HTS platform that can be used to improve antiviral drug discovery. Infection by Dengue virus causes ~100 million illnesses annually, with outbreak severity and frequency increasing with time. This is true of all RNA viruses, which includes Zika, Ebola, Influenza, Yellow Fever, and Hepatitis C, to name a few. In addition t o the health burden Dengue alone represents, dengue fever constitutes a large global financial burden (~39 billion USD), and with 20% of the worlds population living in at-risk areas there is an urgent need for dengue therapeutics. There is currently no approved universal vaccine or dengue specific antiviral drug. Current antiviral drugs on the market target viral enzymes; viral enzymes often have structures that are not present in mammalian cells and are absolutely required for their life cycle making them good targets. High throughput screening assays for dengue drug discovery are either performed in vitro or in vivo. In vitro assays are target specific, but results do not translate well when performed in cells. In vivo assays are more clinically relevant, but the target of the drug is unknown, making it hard to predict toxicity and tailor the drug to improve efficacy. Despite interest in dengue antivirals, no dengue antiviral has successfully passed clinical testing, suggesting a need for a better antiviral screening platform that can combine the advantages of in vitro and in vivo assays. We plan to use our propriety RNA aptamer technology to develop a target-specific cell-based HTS platform targeting the RNA-dependent RNA polymerase (RdRP) of dengue virus. RdRP is an ideal target in Dengue due to its high identity across serotypes, and an ideal target in RNA viruses because of its critical role in viral replication, and its lack of a mammalian counterpart. The goal of this application is to develop a fluorescent sensor capable of monitoring RdRP transcription in a cell line stably expressing viral proteins to advance dengue antiviral discovery. Phase II of this application is to develop a completely comprehensive cell line stably expressing both viral proteins and a fluorescent sensor for use in HTS assays. Phase II will also include the development of similar assays for other viruses, including Zika and Ebola, based off design knowledge from this Phase I grant.
Public Health Relevance Statement: Project Narrative RNA viruses, such as dengue virus, infect hundreds of millions of people annually; dengue virus is one of the most prolific RNA viruses, with ~20% of the worlds population in at risk areas. To improve current efforts to find dengue antiviral agents, we propose to develop a robust cell-based assay capable of specifically monitoring viral transcription in cellular conditions. Successful assay development will enhance dengue drug development and design principles can be applied to other RNA viruses.
Project Terms: 3' Untranslated Regions; Address; Antiviral Agents; Antiviral Therapy; aptamer; Aptamer Technology; Area; assay development; base; Binding; Biological Assay; Broccoli - dietary; Cell Line; Cell physiology; Cells; Cellular Assay; Cessation of life; clinically relevant; combat; Dengue; Dengue Fever; Dengue Infection; Dengue Virus; design; Development; Disease Outbreaks; DNA cassette; Drug Design; drug development; drug discovery; Drug Targeting; Ebola; Ensure; Enzymes; Extravasation; Family; Financial Hardship; Flaviviridae; Fluorescence; Frequencies; Genetic Transcription; Goals; Grant; Health; Hepatitis C; high throughput screening; Human; improved; In Vitro; in vitro Assay; in vivo; Industry Standard; Influenza; inhibitor/antagonist; interest; Internal Ribosome Entry Site; Knowledge; Lead; Life Cycle Stages; Luciferases; Mammalian Cell; Measures; Monitor; Names; Nonstructural Protein; Open Reading Frames; Pathway interactions; Permeability; Pharmaceutical Preparations; Phase; Polymerase; Population; Protein Biosynthesis; Publishing; Reporter; research clinical testing; Resources; Risk; RNA; RNA Polymerase Inhibitor; RNA Viruses; RNA-Directed RNA Polymerase; Role; screening; sensor; Serotyping; Severities; Signal Transduction; small molecule; Specificity; Spinach - dietary; stable cell line; Structure; Structure-Activity Relationship; Symptoms; System; Testing; Therapeutic; Time; Toxic effect; Transfection; Translating; Translations; universal vaccine; Vaccines; Viral; Viral Genome; Viral Proteins; viral RNA; Virus; Virus Replication; Yellow Fever; ZIKA
