title: Yeast based small molecule screen for inhibitors of SARS-CoV.
abstract: Severe acute respiratory coronavirus (SARS-CoV) emerged in 2002, resulting in roughly 8000 cases worldwide and 10% mortality. The animal reservoirs for SARS-CoV precursors still exist and the likelihood of future outbreaks in the human population is high. The SARS-CoV papain-like protease (PLP) is an attractive target for pharmaceutical development because it is essential for virus replication and is conserved among human coronaviruses. A yeast-based assay was established for PLP activity that relies on the ability of PLP to induce a pronounced slow-growth phenotype when expressed in S. cerevisiae. Induction of the slow-growth phenotype was shown to take place over a 60-hour time course, providing the basis for conducting a screen for small molecules that restore growth by inhibiting the function of PLP. Five chemical suppressors of the slow-growth phenotype were identified from the 2000 member NIH Diversity Set library. One of these, NSC158362, potently inhibited SARS-CoV replication in cell culture without toxic effects on cells, and it specifically inhibited SARS-CoV replication but not influenza virus replication. The effect of NSC158362 on PLP protease, deubiquitinase and anti-interferon activities was investigated but the compound did not alter these activities. Another suppressor, NSC158011, demonstrated the ability to inhibit PLP protease activity in a cell-based assay. The identification of these inhibitors demonstrated a strong functional connection between the PLP-based yeast assay, the inhibitory compounds, and SARS-CoV biology. Furthermore the data with NSC158362 suggest a novel mechanism for inhibition of SARS-CoV replication that may involve an unknown activity of PLP, or alternatively a direct effect on a cellular target that modifies or bypasses PLP function in yeast and mammalian cells.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland, United States of America.
authors.forename: Matthew
authors.initials: M
authors.lastname: Frieman
authors.forename: Dipanwita
authors.initials: D
authors.lastname: Basu
authors.forename: Krystal
authors.initials: K
authors.lastname: Matthews
authors.forename: Justin
authors.initials: J
authors.lastname: Taylor
authors.forename: Grant
authors.initials: G
authors.lastname: Jones
authors.forename: Raymond
authors.initials: R
authors.lastname: Pickles
authors.forename: Ralph
authors.initials: R
authors.lastname: Baric
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Engel
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D015195:Drug Design; D004847:Epithelial Cells / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D009975:Orthomyxoviridae / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000821:virology; D013678:Technology, Pharmaceutical / Q000379:methods; D014132:Trachea / Q000378:metabolism; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents; D003470:Culture Media; D049452:Green Fluorescent Proteins