Abstract
SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allows for rapid movement of the existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites by TMPRSS2. Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.
Keywords: COVID-19; TMPRSS2; antiviral; drug repurposing; high-throughput screening.
【저자키워드】 COVID-19, Drug repurposing, Antiviral, high-throughput screening., TMPRSS2, 【초록키워드】 Drug repurposing, SARS-CoV-2, antiviral therapy, ACE2, Vaccine, mass spectrometry, clinical trial, therapy, Antiviral, COVID-19 pandemic, clinical trials, protease, viral entry, Spike protein, approved drugs, Receptor-binding domain, Japan, cleavage, nafamostat, camostat, antiviral therapeutics, Therapies, Protease activity, followed by, symptom onset, approved drug, biochemical, Compound, candidate, effective antiviral treatment, human clinical trials, fluorescent, molecule, viral pathogen, ACE2-expressing cells, driving, pseudotyped, Prevent, human clinical trial, otamixaban, PCI-27483, biochemical assay, bind, tested, the spike protein, the receptor-binding domain, inhibiting, ACE2-expressing cell, inhibitors of TMPRSS2, Initially, 【제목키워드】 repurposing, drug, Screening, candidate, Potential,