Segmented negative-sense, single-stranded RNA viruses (sNSVs), which include bunyaviruses, are causative agents of human diseases. Rift Valley Fever Virus (RVFV), a bunyavirus, causes hemorrhagic fever in humans with a case fatality rate of patients developing hemorrhagic fever reaching approximately 50% and has been classified by the NIAID as a Category A Priority Pathogen. RVFV is mosquito-borne, but is also capable of using a wide range of insect vectors with potential to spread to Europe and the Americas. RVFV has the potential to cause significant global health and economic impact. Unfortunately, there are no FDA-approved drugs or vaccines for the treatment of the RVFV infection. Therefore, there is an urgent medical need for more potent therapeutics tailored for RVFV. The overall goal of this project is to identify and develop small molecule prophylactics and/or therapeutics for RVFV infections and preferably also for infections of other highly related bunyaviruses. The strategy is to address the unmet medical need by identifying small molecule inhibitors targeting the enzymatic activity of the essential RVFV endonuclease, which exhibits significant structural similarity to other sNSV endonucleases. The approach is to leverage the team's experience with bunyaviral endonucleases and a homogeneous FRET-based biochemical assay to identify small molecules that inhibit the enzymatic activity of bunyaviral endonucleases. In Preliminary Studies, we developed a FRET-endonuclease activity (FRET-EA) assay for RVFV endonuclease and applied the assay in a low/medium-throughput format with Z'-factors â¥0.9. The FRET assay confirmed the inhibition of an FDA approved antiviral to treat Influenza virus (IAV), Baloxavir acid (BXA), against IAV endonuclease. In Phase I, for Aim 1, the FRET-EA assay will be optimized for high-throughput screening. Biochemical and cellular secondary assays, including FRET, thermal shift, and cell-based infectious assays, will be optimized to further evaluate confirmed hits. In Aim 2, the FRET- EA HTS will be applied to diverse chemical libraries of â¥250,000 small molecules for the identification and confirmation of small molecules that inhibit the enzymatic activity of RVFV endonuclease. In Aim 3, hits identified in Aim 2 will be validated in secondary assays for binding to endonuclease, anti-sNSV spectrum, and potency against infectious RVFV. They will also be prioritized based on their drug-likeness and their ADME properties. In Phase II, we will perform structural based mechanism studies and further chemically optimize priority inhibitors for potency, selectivity, in vitro and in vivo pharmacokinetics properties and evaluate them in animal infection models.
Public Health Relevance Statement: Narrative Rift Valley Fever Virus (RVFV), a Category A Priority Pathogen classified by the NIAID, is a highly pathogenic virus that causes hemorrhagic fever in humans with a lethality rate as high as 50% but with no FDA-approved treatment. This proposal aims to discover novel compounds that inhibit the enzymatic activity of the essential RVFV endonuclease, and thereby, block the infection of RVFV. Such early acting inhibitors could be developed into therapeutic drugs to treat bunyaviral infections.
Project Terms: Acids; Americas; Animals; inhibitor; Antiviral Agents; Antiviral Drugs; Antivirals; anti-viral agents; anti-viral compound; anti-viral drugs; anti-viral medication; anti-viral therapeutic; anti-virals; antiviral compound; antiviral medication; antiviral therapeutic; Biological Assay; Assay; Bioassay; Biologic Assays; Bunyaviridae; Cell Culture Techniques; cell culture; cell cultures; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Disease Outbreaks; Outbreaks; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; endonuclease; Epidemic; Europe; Exhibits; Family; Goals; Viral Hemorrhagic Fevers; hemorrhagic fever; Human; Modern Man; In Vitro; Infection; Influenza; Grippe; Insect Vectors; Isotope Labeling; Life Cycle Stages; Life Cycle; life course; Culicidae; Mosquitoes; Mutation; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; Patients; Pharmacokinetics; Drug Kinetics; Proteins; Publishing; Rift Valley fever virus; Risk; EC 2.7.7.48; RNA Replicase; RNA-Dependent RNA Polymerase; RNA-Directed RNA Polymerase; RNA Viruses; Running; Specificity; chemical structure function; structure function relationship; Structure-Activity Relationship; Vaccines; Vesicular stomatitis Indiana virus; VSV; Vesicular Stomatitis Virus; Viral Genome; virus genome; Virus Diseases; Viral Diseases; viral infection; virus infection; virus-induced disease; Virus Replication; viral multiplication; viral replication; virus multiplication; Virus; Measures; Livestock; Farm Animal; base; Phase; Biochemical; Medical; Series; Chemicals; Blood Serum; Serum; African; Measurement; anti-viral therapy; viral infectious disease treatment; Antiviral Therapy; Collaborations; Case Fatality Rates; treatment vaccines; vaccine for the treatment; vaccine for treatment; therapeutic vaccine; Therapeutic; Therapeutic Agents; Knowledge; prophylactic; System; Country; Viral; Lytotoxicity; cytotoxicity; experience; nucleoside analog; FRET; Förster Resonance Energy Transfer; Fluorescence Resonance Energy Transfer; chemical library; small molecule libraries; epizootic; Toxicities; Toxic effect; Structure; novel; economic impact; Modeling; Sampling; Property; High Throughput Assay; high throughput screening; drug discovery; Pathogenicity; Molecular Interaction; Binding; small molecule; Influenza Virus; influenzavirus; Address; global health; Phlebovirus; sandfly fever virus group; Genus Phlebovirus; Affinity; Mammalian Cell; NIAID; National Institute of Allergy and Infectious Disease; RNA Polymerase Inhibitor; in vivo; Validation; Development; developmental; N-terminal; NH2-terminal; innovation; innovate; innovative; human disease; FDA approved; counterscreen; lead series; Drug Targeting; small molecule inhibitor; Orthobunyavirus; Bunyavirus; pathogenic virus; viral pathogen; virus pathogen; Category A pathogen; Category A priority pathogen; mosquito-borne; mosquitoborne
