Protocol for in vitro fluorescence assay of papain-like protease and cell-based immunofluorescence assay of coronavirus infection

Here, we describe detailed steps to constitute an in vitro assay for monitoring papain-like protease of coronavirus and a cell-based immunofluorescence infection assay. These assays can be adapted for high-throughput screen to determine the efficacy of novel protease inhibitors of coronaviruses and other viruses. In addition, cell-based immunofluorescence infection assay can be used to visually analyze antiviral efficacy of any novel compounds. Graphical Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses.

The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors. Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection.

Chemical screen uncovers novel structural classes of inhibitors of the papain-like protease of coronaviruses

The papain-like protease (PLpro) of coronaviruses is an attractive antiviral target to inhibit both viral replication and interference of the host immune response. We have identified and characterized three novel classes of small molecules, thiophene, cyanofuran, and triazoloquinazoline, as PLpro inhibitors Thiophene inhibited the PLpro of two major coronaviruses, Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) including SARS-CoV-2, while cyanofuran and triazoloquinazoline more selectively inhibited MERS-CoV PLpro. Unlike GRL0617, a known PLpro inhibitor, all three compounds contain no naphthyl group but like GRL0617 were predicted to fit on the cleft near the BL2 loop. Docking studies further revealed that the location and direction of the binding determined their specificity to different coronaviruses. Together, our work demonstrates that the BL2 loop and nearby regions are outstanding druggable targets, and our three inhibitors can be applicable to the development of therapeutics for coronavirus infection. Graphical

Raloxifene as a treatment option for viral infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused corona virus disease 2019 (COVID-19) pandemic and led to mass casualty. Even though much effort has been put into development of vaccine and treatment methods to combat COVID-19, no safe and efficient cure has been discovered. Drug repurposing or drug repositioning which is a process of investigating pre-existing drug candidates for novel applications outside their original medical indication can speed up the drug development process. Raloxifene is a selective estrogen receptor modulator (SERM) that has been approved by FDA in 1997 for treatment and prevention of postmenopausal osteoporosis and cancer. Recently, raloxifene demonstrates efficacy in treating viral infections by Ebola, influenza A, and hepatitis C viruses and shows potential for drug repurposing for the treatment of SARS-CoV-2 infection. This review will provide an overview of raloxifene's mechanism of action as a SERM and present proposed mechanisms of action in treatment of viral infections.