Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.

<i>In silico</i> studies on novel inhibitors of MERS-CoV: Structure-based pharmacophore modeling, database screening and molecular docking

“…Treatment with interferon has also been suggested as a promising therapeutic strategy against MERS-CoV [6]. In 2013, first generation of protease inhibitor antiviral drugs, such as N3 [3] and CE-10 [7] that were optimized for inhibition of SARS-CoV 3CLpro have been reported to block the activity of MERS-CoV 3CLpro.…”

Middle East respiratory syndrome: obstacles and prospects for vaccine development

“…Polyproteins pp1a and pp1ab; proteolytically processed to Nonstructural proteins (Nsp) 1–16 [139,140]…”

The Planning, Execution, and Evaluation of a Mass Prophylaxis Full-Scale Exercise in Cook County, IL

“…2,3 Immunizations and antiviral drugs are being developed and tested against these pathogens, and if these viruses or future emergent viruses were to become pandemic and be transmitted to the United States, systems must be in place to administer either vaccines or antivirals to susceptible individuals. 3,4 These recent outbreaks, taken with the continued threat of bioterrorism from either foreign or domestic entities, highlight the importance of detailed planning and execution of training exercises designed to dispense prophylactic medication to a local population. 1,[5][6][7] In the event of a bioterrorism attack or communicable disease outbreak, mass prophylaxis is one of the most critical response mechanisms and is a major focus of public health preparedness and funding.…”