Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.
Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC(50)s) of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (50% effective concentration [EC(50)] = 8.95 μM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC(50)] = >250 μM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target.
Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA.
Adeyemi O
AO
Adedeji
Kamalendra
K
Singh
Nicholas E
NE
Calcaterra
Marta L
ML
DeDiego
Luis
L
Enjuanes
Susan
S
Weiss
Stefan G
SG
Sarafianos
D000998:Antiviral Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004265:DNA Helicases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000037:antagonists & inhibitors* / Q000235:genetics; D012367:RNA, Viral / Q000037:antagonists & inhibitors* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D045169:Severe Acute Respiratory Syndrome / Q000188:drug therapy / Q000821:virology; D054852:Small Molecule Libraries / Q000494:pharmacology; D014230:Triazoles / Q000494:pharmacology*; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; C000601132:SSYA10-001; D054852:Small Molecule Libraries; D014230:Triazoles; D014764:Viral Proteins; D004265:DNA Helicases