COVID-19: Assay Development, HTS and Drug Repurposing to identify potential therapeutics against SARS-CoV-2

National Center For Advancing Translational Sciences

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Abstract

During this past reporting period, NCATS continued development of a number of target-based SARS-CoV-2 assays to screen for potential hits against the virus. Optimized assays were screened against a panel of drug repurposing, clinical candidate and antiviral libraries that have been assembled at NCATS. These results have been made freely available to public with unprecedented speed through the NCATS OpenData Portal and through a number of preprint/journal publications. These assays include both virus (e.g. Spike protein) and host (e.g. ACE2, TMPRSS) targets, as well as BSL3 live SARS-CoV-2 virus infectivity assays accessed through research contracts. Many assays have been solely developed in-house at NCATS, while a number of others have been designed in collaboration with both intramural and extramural laboratories with subject area expertise. The COVID-19 pandemic is a pressing public health emergency garnering rapid response from scientists across the globe, and NCATS scientists have been conducting screening and drug repurposing against a number of SARS-CoV-2-related targets in response: 1) Host cell invasion is initiated through direct binding of the viral spike protein to the host receptor ACE2. Members of this team developed a proximity-based AlphaLISA assay to measure binding of SARS-CoV-2 spike RBD protein to ACE2 and used it to run a drug-repurposing screen, yielding 25 high-quality, small-molecule hits worthy of follow-up in cell-based models. This established AlphaLISA RBD-ACE2 platform allows scientists to evaluate disruption of this viral-host interaction, and this assay has been reported at multiple conferences. 2) The TMPRSS2 enzyme has been shown to be critically important for priming SARS-CoV-2 for viral entry into host cells, yet no SARS-CoV-2-related biochemical assay for this target had been previously reported. To enable inhibitor discovery and profiling of FDA-approved therapeutics, this team developed a biochemcial assay using recombinant TMPRSS2 suitable for high-throughput screening applications. The team validated this important assay, and demonstrated effectiveness to quantify inhibition down to subnanomolar concentrations using clinically approved proteases. This TMPRSS2 work will enable further screening against this important antiviral host target, and provide insight into selectivity and potency of repurposing candidates. 3) Antibodies that physically block viral entry of SARS-CoV-2 may provide leads for further therapeutic development. This project team used an innovative combination of humanized nanobody library and enrichment strategies (also known as phage panning) to discover several clones of antibodies capable of high-affinity binding to SARS-CoV-2 spike protein. Because these highly engineered antibody variants already incorporate beneficial features such as similarity to human antibodies, they are expected to be developed into viable therapeutic leads faster than other types of neutralizing antibodies. 4) Pulmonary fibrosis is a major problem associated with severe cases of SARS-CoV-2 infections and the leading cause of mortality associated with COVID-19. Currently, the only treatment is supplemental oxygen and mechanical ventilation to hospitalized COVID patients. There is no effective therapeutic measure to mitigate the disease. The goal of this project is to develop small molecular inhibitors as therapeutic measures to mitigate COVID-19 associated lung fibrosis.The project's goal is to develop small molecular inhibitors as therapeutic measures to mitigate COVID-19 associated lung fibrosis. During this period, we tried to optimize the absorbance-based assay developed in our collaborator's lab and develop fluorescence-based thrombin activity assay.