A biophysical assay targeting SARS CoV-2 RNA

PROJECT SUMMARY The world is immersed in a health crisis rivaled only by the Spanish flu pandemic of 1918-1919. The difference between the crisis of today and the crisis a century ago is that we have advanced communication technology greatly so that huge populations of people around the globe are aware of risks and can take appropriate precautions, such as the employment of quarantining, isolation, social distancing, and masks and handwashing. Governments have closed schools and outlawed large social gatherings. Medical care has greatly improved, and those most affected have received symptomatic treatment in the absence of a cure for CoVID-19. The problem must be dealt at multiple fronts, such as vaccine development, drug development and new technologies, assays for mitigating viral effects. The proposed project is significant because it proposes a novel in vitro biophysical screening assay for a unique and yet untapped RNA structure in SARS-CoV-2 virus, that can be used in the future to generate RNA specific antiviral compounds. Nucleic acids are promising avenues for drug design, both as therapeutics and as targets. Here we propose an innovative screening approach for identification of a novel class of ligands that are specific for an RNA element within the viral RNA genome that is vital for replication of the virus, and we propose a biophysical screening assay as a first step for identifying such ligands. First, as outlined in Specific Aim 1, we will characterize a model nucleic acid (RNA) domain that will be synthesized, characterized and used to identify a RNA specific fluorescent probe. The optimized probe will then be used for developing a high-throughput screening assay for discovery of nanomolar binders to this RNA. The RNA specific high-affinity binders will then be combined with sequence-specific RNA binding ligands to validate the assay development and its utility. The mechanism of action will be confirmed using inhibition of firefly luciferase translation in a reporter gene assay (Specific Aim 2). While the focus of this application, as the needed first step, is on the development and validation of the biophysical assay for the underlying SARS-CoV-2 RNA structures, a successful validation of the approach will open the doors for discovery and development of lead compounds for inhibition of SARS-CoV in Phase II studies. NUBAD and its team of scientists and collaborators is uniquely qualified to perform these assays and develop the potential leads in Phase II, in addition to providing a template for the scientific community to use the screening technology for their own discovery platforms. Success of the approach will also allow us to expand the screening technology to other RNA structures in SARS and other RNA viruses, and provide the screening resource as a service to the scientific community.