SARS-CoV Replication Inhibitor that Interferes with the Nucleic acid Unwinding of the Viral Helicase.

Abstract:

Severe acute respiratory syndrome (SARS) is a highly contagious disease caused by SARS-associated 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 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 (ds) RNA and dsDNA unwinding activities of nsp13 with an IC(50)s of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (EC(50)=8.95 μM) in a SARS-CoV replicon assay with low cytotoxicity (CC50= >250 μM), suggesting that the helicase plays a yet unidentified critical role in SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a non-competitive inhibitor of nsp13 with respect to the 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 HCV helicase or other bacterial and viral RNA-dependent RNA polymerases and 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 acid or ATP. Hence, it is possible that SSYA10-001 inhibits nsp13 unwinding 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 SARS-CoV life cycle, which could be a model for other nidoviruses, and also a candidate for further development as a SARS antiviral.

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