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

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.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Cell-based membrane fusion assay for the MERS-S protein using the dual split protein (DSP) reporter. Membrane fusion mediated by the MERS-S protein was monitored by coculturing the effector and target cells expressing the respective split reporter proteins (dual split proteins [DSPs]). DSPs are split chimeric proteins of GFP and Renilla luciferase (RL) and recover both GFP (fluorescence) and RL (luminescence) signals by their reassociation upon mixture of cellular contents during membrane fusion. The effector cells express MERS-S protein, and the target cells express CD26 and TMPRSS2. DSP1-7 and DSP8-11, DSPs that reassociate themselves; S, the S protein of MERS-CoV; N, nucleus.

FIG 2

Required components for membrane fusion mediated by MERS-S. The effector 293FT cells expressing DSP8-11 were transduced with MERS-S. The target 293FT cells expressing DSP1-7 were transduced with CD26 and TMPRSS2 separately or simultaneously. Different combinations of these effector and target cells were cocultured, and the resulting RL activity was measured (shown in relative light units [RLU]). The names of the transduced proteins are indicated below the bar graph (None, no proteins other than the DSP reporters; Cont, control).

FIG 3

The results of the high-throughput screening (HTS) of 1,017 FDA-approved drugs in the DSP assay for MERS-S. Results of screening of 1,017 drugs using the HTS DSP assay are shown. (A) The vertical axis shows the reading of the DSP activity (RL activity) for the tested drugs (1 μM). The RL values were normalized to the control, which contained DMSO only. The horizontal axis shows the identification number arbitrarily assigned to each drug. Each dot represents an individual drug. The dotted line indicates 20% of the control value. The value determined for the most active drug, nafamostat (1.66% of the control value), is indicated with a red arrow and red dot. (B) The effect of each drug on RL activity itself. To rule out the nonspecific direct inhibitory effect of each drug on the RL activity itself, each drug (1 μM) was applied to the cells coexpressing both DSP1-7 and DSP8-11, which harbored reassociated active DSP. The results are shown as in panel A. The value for nafamostat (106.1% of the control value) is indicated with a red arrow and red dot.

FIG 4

Effects of several serine protease inhibitors on MERS-S-mediated membrane fusion, as examined by the DSP assay. Several clinically used serine protease inhibitors were evaluated by the DSP assay for their effects on MERS-S-mediated membrane fusion. (A) The effect of each drug on the coculture fusion assay using DSP as a reporter. Drugs were tested at different concentrations, and the additional proteins other than reporters (DSPs) transduced into the effector and target cells are indicated below the graph. none, no additional protein. The relative cell fusion value is represented as the DSP value (RL activity measured in RLU) normalized to that of the control assay performed with DMSO alone. Gabe, gabexate mesylate; Nafa, nafamostat; Camo, camostat mesilate; Sive, sivelestat sodium tetrahydrate; Riva, rivaroxaban; Tela, telaprevir; Ulina, ulinastatin; Sime, simeprevir. (B) The effect of each drug on RL measurement. Each drug was added to cells coexpressing DSP1-7 and DSP8-11 to evaluate its direct inhibitory effects on RL. The relative DSP signal is indicated in the vertical axis by setting the control value with DSP alone as 100%. (C) The DSP assay system using Calu3 cells as target cells. DSP1-7 was constitutively expressed in Calu3 cells. Using 293FT-derived effector cells expressing DSP8-11 and MERS-S (indicated as MERS-S), the DSP assay was performed in the presence of nafamostat or camostat. Effector cells expressing only DSP8-11 (indicated as “None”) were used as a control.

FIG 5

Inhibition of MERS-CoV infection by nafamostat in vitro. Data represent the results of inhibition of viral entry by administration of the serine protease inhibitors camostat and nafamostat. (A) Calu3 cells pretreated with different concentrations of individual inhibitors were challenged with MERS-CoV (EMC2012) at an MOI of 10, and the amount of internalized viral RNA was quantified by real-time PCR. The figure is representative of the results of duplicate experiments. The asterisks indicate the statistically significant differences determined by Student's t test (*, P < 0.001; **, P < 0.01; ***, P < 0.05). n.s., no significant difference. (B) Effects of inhibitors on viral replication. After viral infection, inhibitors were added at different concentrations, and the amount of progeny viruses released into the culture medium was quantified using Vero/TMPRSS2 cells. The figure shows representative results from day 1. Statistical analysis was performed using Student's t test (**, P < 0.01). n.s., no significant difference.