Testing of Middle East respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry.

As of 23 July 2014, 837 laboratory-confirmed cases of Middle East respiratory syndrome (MERS-CoV) infection, including 291 deaths, had been reported to the WHO (http://www.who.int /csr/disease/coronavirus_infections/en/), raising concerns about its pandemic potential and calling for the development of vaccines and therapeutics against MERS-CoV infection. We previously identified peptidic HIV-1 and severe acute respiratory syndrome coronavirus (SARS-CoV) fusion inhibitors (1, 2), which led to the development of MERS-CoV spike (S) protein-mediated cell-cell fusion and six-helix bundle (6-HB) formation assays. Using these assays, we identified a peptide from the MERS-CoV S protein HR2 region, termed HR2P, that inhibited 6-HB formation, cell-cell fusion, and MERS-CoV replication (3). To identify small-molecule MERS-CoV fusion inhibitors, we used a cell-cell fusion assay to screen 1,280 compounds from an FDAapproved drug library obtained from MicroSource Discovery Systems, Inc. (Gaylordsville, CT), but none of the compounds at 10 M could significantly inhibit MERS-CoV S-mediated membrane fusion. Most recently, de Wilde et al. (4) and Dyall et al. (5) used a cytopathogenic effect assay to screen several hundred compounds from FDA-approved drug libraries and identified a series of compounds that inhibit the replication of both MERS-CoV and SARSCoV in the low micromolar range. Although the mechanisms of action have not been defined, both groups suggested that some of the drugs, such as chlorpromazine (a clathrin-mediated endocytosis inhibitor), might block virus entry (4, 5). Coronavirus enters the target cell via endocytosis or plasma membrane fusion, while the latter is the main pathway for MERSCoV entry (3). To determine whether these reported MERS-CoV replication inhibitors also block virus entry via plasma membrane fusion, we tested 16 compounds with MERS-CoV replication-inhibiting activity available in the FDA-approved drug library from MicroSource and ribavirin and mycophenolic acid (Sigma-Aldrich), which were reported to inhibit MERS-CoV replication (6), for inhibitory activity on MERS-CoV S-mediated cell-cell fusion using HR2P as a control. A cell-cell fusion inhibition assay was performed as we described before (3). Briefly, Huh-7 cells were used as target cells and 293T cells, which simultaneously express MERS-CoV S protein and enhanced green fluorescent protein (293T/MERS/EGFP), were used as effector cells. 293T/MERS/ EGFP cells were cocultured with HR2P or compounds at graded concentrations (initial concentration of 40 M) for 30 min, added to Huh-7 cells, and then incubated at 37°C for 2 to 4 h (3). As expected, HR2P inhibited cell-cell fusion with an IC50 (half-maximal inhibitory concentration) of 1 M and effectively blocked 6-HB formation. In contrast, most of these compounds exhibited no significant inhibitory activity at 40 M, except for the three neurotransmitter inhibitors (chlorpromazine, promethazine, and fluphenazine), which were moderate inhibitors of cell-cell fusion with IC50s of about 20, 20, and 29 M, respectively (Table 1). Subsequently, we determined the inhibitory activities of these compounds (40 M) on 6-HB formation between HR1P and HR2P-fluorescein isothiocyanate (FITC) by using a fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) assay adapted from the FN-PAGE assay for testing of HIV fusion inhibitors (7). As expected, HR1P showed no band because it carries net positive charges, thus migrating up and off the gel under native electrophoresis conditions, which is consistent with the results of HR1 peptides from HIV-1 (7) and SARS-CoV (2), while HR2PFITC showed a band at a lower position. The mixture of HR1P and HR2P-FITC showed a band at a higher position, suggesting the formation of an HR1P/HR2P-FITC complex, possibly the 6-HB band (Fig. 1). In the presence of HR2P, the upper band disappeared while the lower HR2P-FITC band was displayed, suggesting that HR2P binds to HR1P and blocks 6-HB formation between HR2P-FITC and HR1P. However, none of the MERS-CoV replication inhibitors at 40 M could block 6-HB formation by HR2P-FITC and HR1P (Fig. 1 and Table 1). The cytotoxicity of these MERS-CoV replication inhibitors to the Huh-7 cells that were used as target cells in the cell-cell fusion assay was determined by using cell counting kit 8 (Dojindo, Kumamoto, Japan) as previously described (3). Except for emetine dihydrochloride, triflupromazine hydrochloride, and clomipramine hydrochloride with CC50s (the concentration of a compound causing 50% cytotoxicity) of 28.63, 33.58, and 5 M, respectively, none of the compounds exhibited cytotoxicity at 40 M (Table 1). We then tested the inhibitory activity of the 16 MERS-CoV replication inhibitors on MERS-CoV pseudovirus-based, clathrin-mediated endocytosis by using an assay adapted from the method for testing of SARS-CoV inhibitors as previously described (8). The pseudotyped MERS-CoV was constructed as describe before (3). Huh7 cells were incubated with chlorpromazine hydrochloride (as a positive control) and other MERS-CoV replication inhibitors at graded concentrations for 1 h and then infected with the MERS-CoV pseudovirus for an additional 12 h. After extensive washes with phosphate-buffered saline to remove