Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay

Abstract

e Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

“…In this context, it is noteworthy that the serine protease inhibitor camostat mesylate, which blocks TMPRSS2 activity (Kawase et al, 2012;Zhou et al, 2015), has been approved in Japan for human use, but for an unrelated indication. This compound or related ones with potentially increased antiviral activity (Yamamoto et al, 2016) could thus be considered for off-label treatment of SARS-CoV-2-infected patients.…”

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

“…Expression of TMPRSS2 at the cell surface induces both virus entry into cells and cell-cell fusion of S protein-expressing cells (4,10,27). Furthermore, these phenomena are suppressed by the serine protease inhibitors camostat mesylate and nafamostat mesylate (4,10,28). In addition, other extracellular proteases, such as trypsin and elastase, activate the MERS-CoV S protein in a manner similar to that of TMPRSS2 (10).…”

TMPRSS2: A potential target for treatment of influenza virus and coronavirus infections

“…In addition, camostat at a concentration of 10 mM impairs MERS-CoV entry 15-fold in Vero-TMPRSS2 cells, and the amount of viral RNA in the culture supernatant of Calu-3 cells is reduced 270-fold in the presence of 100 mM camostat at three days post MERS-CoV infection [22]. More recently, nafamostat, a serine protease inhibitor, was reported, to block MERS-CoV infection in vitro via inhabiting the activity of TMPRSS2, and reduced viral entry by 100-fold at a concentration of as low as 1 nM, which is more effective than camostat [74].…”