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.

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.

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.

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.