Evaluation of SSYA10-001 as a Replication Inhibitor of Severe Acute Respiratory Syndrome, Mouse Hepatitis, and Middle East Respiratory Syndrome Coronaviruses

Abstract

We have previously shown that SSYA10-001 blocks severe acute respiratory syndrome coronavirus (SARS-CoV) replication by inhibiting SARS-CoV helicase (nsp13). Here, we show that SSYA10-001 also inhibits replication of two other coronaviruses, mouse hepatitis virus (MHV) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). A putative binding pocket for SSYA10-001 was identified and shown to be similar in SARS-CoV, MERS-CoV, and MHV helicases. These studies show that it is possible to target multiple coronaviruses through broad-spectrum inhibitors.

TEXT

Coronaviruses are enveloped positive-sense RNA viruses that cause a range of diseases in humans and animals. The present report focuses on three highly pathogenic coronaviruses, two of which infect humans. Severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for the life-threatening viral respiratory illness known as SARS, which emerged from Southern China in November 2002 and spread to other parts of the world, including North America, South America, and Europe (1, 2). Middle East respiratory syndrome CoV (MERS-CoV) is a newly discovered coronavirus that caused severe pneumonia in patients in the Middle East (Saudi Arabia, Jordan, Qatar, and the United Arab Emirates), Europe (the United Kingdom, France, Italy, and Germany), North Africa (Tunisia and Egypt) (3), and the United States of America. As of 13 May 2014, WHO listed 538 laboratory-confirmed cases of MERS-CoV infections worldwide, including 145 deaths (http://www.cdc.gov/media/releases/2014/p0512-US-MERS.html). Mouse hepatitis virus (MHV) is a murine coronavirus that can cause a wide range of illnesses in mice depending on the viral strain and the route of infection; these include respiratory, gastrointestinal, hepatic, and central nervous system (CNS) diseases (4). The MHV-A59 strain used in this study is a neuropathogenic strain. To date, no drugs have been approved for the treatment of any coronavirus infection.

We recently identified various small-molecule inhibitors of SARS-CoV that target various steps of SARS-CoV replication (5–8). Among them was SSYA10-001, a 1,2,4 triazole that prevents the helicase activity of SARS-CoV nsp13 and blocks SARS-CoV replication (8). We were particularly interested in this helicase inhibitor because, unlike entry inhibitors, that target highly variable surface glycoproteins, SSYA10-001 targets the SARS-CoV nsp13 helicase, which shares significant homology with other coronavirus helicases (Fig. 1). Hence, we hypothesized that the binding pocket of SSYA10-001 in SARS-CoV nsp13 is conserved among different coronavirus helicases, raising the exciting possibility of discovering broad-spectrum coronavirus inhibitors.

FIG 1.

Sequence alignment of nsp13/SF1 helicases from α-, β-, and γ-coronaviruses. The dashes represent residues identical to SARS-CoV helicase residues. The stars represent the gap in the sequence. This figure shows six conserved SF1 helicase motifs, ATP hydrolysis active site (highlighted in red) in SARS-CoV (GenBank accession no. AAP13442.1), human CoV (HCoV)-229E (GenBank accession no. AAG48591.1), HCoV-HKU1 (GenBank accession no. AAT98578.1), MHV (GenBank accession no. NP_740617.1), MERS-CoV (GenBank accession no. AFV09327.1), and turkey CoV (TCoV) (GenBank accession no. YP_001941186.1) nsp13 helicases. SSYA10-001 binding pocket residues are highlighted in green. For simplicity, the first approximately 240 N-terminal residues are not shown. The levels of homology between SARS-CoV and the 229E, NL63, HKU1, and TCoV helicases are 76%, 76%, 82%, and 68%, respectively.

To locate the binding site of SSYA10-001 within SARS-CoV nsp13, we used three pocket-prediction programs: “SiteMap” (Schrodinger Suite), “SiteId” (Tripos Associates), and “Q-site finder” (9). This approach identifies binding sites based on volumes roughly equivalent to the ligand volume, in this case, that of SSYA10-001 (9). The putative binding site comprising residues Y277, R507, and K508 was chosen for further evaluation. We used site-directed amino acid substitutions to construct SARS-CoV nsp13 enzymes with any one of the following substitutions: Y277A, R507A, or K508A. Cloning and protein expression of these enzymes were as previously described (8). Two of the three targeted proteins were successfully prepared to high homogeneity (>90%) and in active forms (Fig. 2A). We determined the unwinding activities of wild-type (WT), Y277A, and K508A SARS-CoV nsp13 helicases in the presence of various concentrations (0, 2.5, 5, 10, 25, 50, 75, and 100 μM) of SSYA10-001, using a FRET-based assay as previously described by us (8). The results showed that the Y277A and K508A amino acid substitutions conferred resistance to SSYA10-001, as their estimated respective 50% inhibitory concentrations (IC₅₀s) were 12 and 50 μM, respectively, compared to 5.9 μM for WT SARS-CoV nsp13 (Fig. 2). Therefore, we concluded that Y277 and K508 are part of the binding pocket for SSYA10-001 within SARS-CoV nsp13. Importantly, sequence alignment of several coronavirus helicases revealed that the residues of the proposed inhibitor binding site are largely conserved in multiple coronaviruses (Fig. 1). Hence, we built homology-derived molecular models of MERS-CoV and MHV nsp13 helicases using “Prime” software (for homology-derived molecular models) and “Glide” with extra precision (XP) and “Induced Fit Docking” workflow (for docking), both integrated into “Maestro” of Schrodinger Suite (Schrodinger Inc.) as previously described (10). Comparison of the three modeled pockets revealed significant similarities (Fig. 3) and suggested that SSYA10-001 may also be a potential antiviral for MHV and MERS-CoV.

FIG 2.

Enzymatic activities of nsp13 WT, nsp13 Y277A, and nsp13 K508A in the presence and absence of SSYA10-001. (A) nsp13 WT, nsp13 Y277A, and nsp13 K508A (50 nM) were incubated in the presence of 20 mM HEPES, 20 mM NaCl, 0.01% bovine serum albumin (BSA), 2 mM dithiothreitol (DTT), 5% glycerol, and 5 mM MgCl₂. The helicase reaction was initiated by the addition of 100 nM 31/18-mer (13 single stranded [ss], 18 double stranded [ds]) as the substrate (Cy3 labeled) (8) at 30°C, along with 0.5 mM ATP and a 2 μM concentration of unlabeled single-stranded DNA (ssDNA) with a sequence complementary to that of the unlabeled DNA strand. The reactions were allowed to proceed for 10 min at 30°C, and the reaction was quenched with 100 mM EDTA, 0.2% SDS, and 20% glycerol. The products were separated and analyzed by the use of 6% nondenaturing PAGE. (B) Helicase reactions for nsp13 WT (Δ) and nsp13 Y277A (◼) and nsp13 K508A (○) were performed in the presence of various concentrations of SSYA10-001 inhibitor. The fraction of unwound DNA was plotted against the concentration of the inhibitor, and the data were fitted to a dose-response curve using GraphPad Prism 5.0. Experiments were performed in triplicate in three independent experiments, and error bars represent standard deviations of the results from three independent experiments.

FIG 3.

SSYA10-001 docking in inhibitor binding pockets of SARS-CoV, MERS-CoV, and MHV nsp13 helicase molecular models. Surface representations of molecular models of nsp13 helicases from three coronaviruses are shown. The inhibitor binding sites with docked inhibitor molecules are shown for the three enzymes. The amino acid residues that were experimentally validated in the SARS-CoV enzyme and the equivalent residues in the other enzymes are shown as orange surface areas. The surface area for the rest of the molecules is shown by atom type (gray, carbon; red, oxygen; blue, nitrogen; yellow, sulfur). The equivalent residues in MERS-CoV and MHV helicases are also shown in an orange surface area representation.

To determine the effect of SSYA10-001 on MERS-CoV replication, VeroE6 cells were seeded into 96-well plates (Corning Costar) at 1 × 10⁴ cells per well and cultured overnight at 37°C. Cells were treated with SSYA10-001 at concentrations of 6.25 μM to 200 μM, or with dimethyl sulfoxide (DMSO) as a vehicle control, for 2 h under normal culture conditions. MERS-CoV (Jordan strain) or SARS-CoV (MA15) was then added to each well at a multiplicity of infection (MOI) of 0.1. After 48 h, the supernatants were harvested. Viral load in the supernatants was assessed using a 50% tissue culture infective dose (TCID₅₀) assay as previously described (7). Drug toxicity was assessed by incubating Vero E6 cells in the presence of SSYA10-001 for 48 h, and percent cell survival was determined by using the CellTiterGlo luminescent cell viability assay (Promega, Madison, WI) according to the manufacturer's instructions and was read on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). As shown in Fig. 4, SSYA10-001 inhibits MERS-CoV and SARS-CoV replications with 50% effective concentrations (EC₅₀s) of ∼25 μM (selectivity index = >20) and 7 μM (selectivity index = >71), respectively, as no significant cytotoxicity was observed even at 500 μM (Fig. 4D). To test the susceptibility of MHV-A59 to SSYA10-001, 4 × 10⁴ mouse fibroblast L2 cells were seeded into each well in a 48-well plate. After 24 h, various concentrations (0, 10, 20, 40, and 80 μM) of SSYA10-001 were added to the cells along with the MHV-A59 virus (R13) at an MOI of 0.01. After 24 h, the cells were harvested and a standard plaque assay was performed to analyze the effect of the compound on MHV replication as previously described (11, 12). As shown in Fig. 4C, SSYA10-001 inhibits MHV replication with an EC₅₀ of ∼12 μM.

FIG 4.

Effect of SSYA10-001 on SARS-CoV (A), MERS-CoV (B), mouse hepatitis virus (neuropathogenic strain) (C), and Vero E6 cells (D). Virus titers or percent cell viability is plotted against inhibitor concentrations using GraphPad Prism 5.0. Experiments were performed in triplicate in three independent experiments; error bars represent standard deviations of the results from three independent experiments.

Based on these results, SSYA10-001 is able to inhibit replication of at least three coronaviruses. Although binding of SSYA10-001 has not been demonstrated in MERS-CoV and MHV nsp13, the molecular modeling data suggest that SSYA10-001 can be docked with a comparable Glide score. Based on the similarities among the models of the inhibitor binding sites, we anticipate that other chemically related 1,2,4 triazoles could also bind to this conserved pocket and help in the discovery of anticoronavirus inhibitors. Ongoing studies are focused on in silico screening for the discovery of such inhibitors using the molecular models of these helicases.

In conclusion, we demonstrated through virological, biochemical, and molecular modeling studies that SSYA10-001, a helicase-targeting small-molecule inhibitor of SARS-CoV helicase, has an antiviral effect on multiple coronaviruses by possibly targeting a conserved binding pocket in nsp13. This compound could serve as a lead for the development of effective broad spectrum anticoronavirus drugs.