Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of smallmolecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking of entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S) but not that of HIV-1 pseudotyped with vesicular stomatitis virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that the compounds act by three distinct mechanisms: (i) SSAA09E2 N-[[4-(4-methylpiperazin- 1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, angiotensin converting enzyme 2 (ACE2); (ii) SSAA09E1 [(Z)-1-thiophen-2-ylethylideneamino]thiourea acts later, by blocking cathepsin L, a host protease required for processing of SARS-S duringviral entry; and (iii) SSAA09E3 [N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide] also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L but prevents fusion of the viral membrane with thehost cellular membrane. Our work demonstrates that there are at least three independent strategies for blocking SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.
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