Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

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.

Repurposing Drugs for the Treatment of COVID-19 and Its Cardiovascular Manifestations

“… 52 In another study, a team of investigators developed a fluorogenic high-throughput in vitro screening assay through which a total of 6030 drug repurposing candidates from National Center for Advancing Translational Sciences (NCATS) annotated libraries were evaluated for inhibiting SARS-CoV-2 replication in Calu-3 cells. 53 Next, a suite of subsequent assays, including a mass spectrometry-based detection assay and a cell-based SARS-CoV-2 pseudotyped particle entry assay, was developed to confirm and assess the hits from the biochemical screen, among which camostat, nafamostat, PCI-27483, otamixaban, and 2 peptidomimetic inhibitors of TMPRSS2 were identified.…”

“…inhibitor to treat leukemia and other malignancies Han et al49 and Aman et al91 CyclosporineA calcineurin inhibitor used to prevent organ rejection after transplantation and in certain autoimmune diseases Dittmar et al50 and Sauerhering et al92 Otamixaban A direct factor Xa inhibitor that was investigated for the treatment for acute coronary syndrome Shrimp et al52 and Hempel et al95 SulfasalazineAn anti-inflammatory to treat ulcerative colitis and rheumatoid arthritis Han et al56 DexamethasoneA corticosteroid used to treat inflammation Fava et al57 and The RECOVERY Collaborative Group96 Baricitinib A Janus kinase inhibitor used for the treatment of rheumatoid arthritis and alopecia areata Fava et al57 and Selvaraj et al97 Tacrolimus A calcineurin inhibitor used to prevent organ rejection after transplantation and in certain autoimmune diseases Fava et al57 Zotatifin A selective inhibitor of the eukaryotic translation initiation factor 4A with potential antineoplastic activity Gordon et al25 and Fava et al57 Nintedanib A tyrosine kinase inhibitor used for the treatment of idiopathic pulmonary fibrosis…”

“…Schultz et al46 and Beigel et al89 NiclosamideA medication used to treat tapeworm infections Almeida et al48 and Braga et al90 Lactoferrin A protein found in the milk of mammals with potential antioxidant, antiviral, and antibacterial properties Mirabelli et al51 and Campione et al93 Nafamostat A synthetic serine protease inhibitor, with anticoagulant, antiinflammatory, mucus clearing, and potential antiviral activities Shrimp et al52 and Takahashi et al94 Hoffmann et al,23 Shrimp et al,52 and Hoffmann et al80 Omipalisib A small molecule inhibitor of PI3K (phosphoinositide 3-kinase) with potential antineoplastic activity Klann et al36 and Jang et al47 SorafenibA protein kinase inhibitor used to treat late-stage kidney, liver, and thyroid cancer Klann et al36 and Meyer et al83 ObatoclaxAn experimental Bcl-2 (B-cell lymphoma 2) inhibitor used to treat leukemias, lymphomas, myelofibrosis, and mastocytosisPatten et al42 and Mao et al85 …”

A Tale of Two Proteases: MPro and TMPRSS2 as Targets for COVID-19 Therapies

“…In clinical trials, it has shown to be safe but not as efficient as the control used. It has reappeared again as a drug of interest because of its ability as a TMPRSS2 inhibitor and hence a potential COVID-19 therapy [ 61 ].…”

Impact of TMPRSS2 Expression, Mutation Prognostics, and Small Molecule (CD, AD, TQ, and TQFL12) Inhibition on Pan-Cancer Tumors and Susceptibility to SARS-CoV-2

“…Hoffmann et al first demonstrated that the SARS-CoV-2 S protein is primed via the TMPRSS2 protease, indicating that TMPRSS2 inhibitors may block viral invasion [ 9 , 12 ], SARS-CoV-2 entry, and coronavirus disease 2019 (COVID-19) outcomes [ 13 ]. These findings suggest that the inhibition of TMPRSS2 expression may protect against cancer progress as well as SARS-CoV-2 invasion.…”