Discovery of Bunyaviral Endonuclease Inhibitors for Antiviral Therapy

Abstract
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 ≥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 ≥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.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.