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

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

During this past reporting period, NCATS worked towards completing a number of SARS-CoV-2 assays, including target-based and live virus assays, to screen for potential hits against SARS-CoV-2. 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. The COVID-19 pandemic continued to be a pressing public health emergency garnering rapid response from scientists across the globe, and NCATS scientists have concluded screening and drug repurposing against a number of SARS-CoV-2-related targets in response. Completed screens are listed below as (1-4), and screening in development is listed in (5). 1) SARS-CoV-2 entry into target cells 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 lung 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 biochemical 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 SARS-CoV-2 entry into host cells (i.e. antibody-mediated viral neutralization) 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 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 a fluorescence-based thrombin activity assay. 5) The National Cancer Institutes (NCI) Natural Products Repository is one of the largest natural products representing a rich and highly diverse set of bioactives in fractionated extracts that are amenable for high throughput screening. Currently there are over 500,000 partially purified natural product fractions available through this collection, with over 1,000,000 partially purified natural product fractions planned. This library has previously not been explored for anti-replication activity against SARS-CoV-2. To enable screening of the NCI Natural Products Repository, we have optimized a previously developed firefly luciferase SARS-CoV-2 live virus assay, as well as have developed a secondary no-wash, Mcherry SARS-CoV-2 assay that enables both toxicity and infection reads in the same well. In addition, we have built a dedicated BSL-3 high throughput screening facility for automated screening of antivirals against SARS-CoV-2 and other pathogens. The NCATS APP BSL-3 HTS Platform is a fully-automated, robotic high throughput screening in 384/1536w assay plates for high containment pathogens (e.g. SARS-CoV-2) that was built inside a BSL-3. This NCATS Installed Enclosure Design Filtration Microzone (DFMZ) contains a customized HTS robotics platforms including, 90 ft2; 540 ft3 (interior space) that is rated for BSL3 level pathogens due to the presence of a Class II Automation Enclosure, inward continuous unidirectional airflow, HEPA- filtered, and minimu34m ISO 5 conditions within the work zone. In addition, the automated platform has necessary components for compound library dispensing including an Echo 655 acoustic dispenser, a V-spin centrifuge, a PlateLoc sealer, and a X-peel peeler, as well as integrated liquid handling including a BioTek EL406 and Certus Flex, and components for integrated screening including a Cytomat incubator which can hold 200 HTS cell plates and a multimode reader, the BMG Pherastar, which equipped with a stacker and a range of filters for multi-assay formats, and an integrated robotics arm.