the virus [9], highlighting the rapid responses of the scientific community to this previously unknown global pathogen. Advancements in synthetic organic chemistry, molecular biology, and informatics have made possible the in vitro and in vivo [10-13]. The term "chemical genetics" has been coined to signify the use of chemicals to per-1 Department of Microbiology 2 Department of Biochemistry turb systematically, and thus determine, the function of proteins in the same way that mutations are used in classical genetics [14-16]. We decided to dissect the pathogenic pathways of the SARS-CoV using chemical Pokfulam, Hong Kong SAR China genetics. We hypothesized that by using forward chemical genetics [15, 16], in which small molecules that in-4 The Aaron Diamond AIDS Research Center 455 First Avenue duce altered phenotypes in cells or organisms are identified and their cellular targets will then be determined New York, New York 10016 subsequently, we will be able to isolate novel small-molecule compounds perturbing the biological pathways that are essential for the pathogenesis of SARS-CoV. Summary The severe acute respiratory syndrome-associated tion with ACE2 [9]. After being internalized into the target coronavirus (SARS-CoV) infected more than 8,000 cells, SARS-CoV undergoes a very rapid replication cypeople across 29 countries and caused more than 900 cle through a series of concerted transcriptional, translafatalities. Based on the concept of chemical genetics, tional, posttranslational, and proteolytic processing we screened 50,240 structurally diverse small moleevents, leading to maturation and release of infective cules from which we identified 104 compounds with viral particles into the culture supernatant [17-19]. We anti-SARS-CoV activity. Of these 104 compounds, 2 speculated that the various biological pathways intarget the SARS-CoV main protease (M pro ), 7 target volved in viral pathogenesis could be perturbed by small helicase (Hel), and 18 target spike (S) protein-angiomolecules using chemical genetic approaches. To extensin-converting enzyme 2 (ACE2)-mediated viral enamine the feasibility of employing chemical genetic try. The EC 50 of the majority of the 104 compounds approaches in SARS-CoV research, we acquired a determined by SARS-CoV plaque reduction assay chemical library (ChemBridge Corporation) of 50,240 were found to be at low micromolar range. Three sestructurally diverse small-molecule compounds that lected compounds, MP576, HE602, and VE607, valivary in functional groups and charges. As the SARSdated to be inhibitors of SARS-CoV M pro , Hel, and viral CoV replicates effectively in Vero cells (African green entry, respectively, exhibited potent antiviral activity monkey kidney cell line) and full cytopathic effects (CPE) (EC 50 Ͻ 10 M) and comparable inhibitory activities in of the infected cells can be observed within 96 hr postintarget-specific in vitro assays. fection, Vero cell CPE was used as a phenotypic indicator of successful viral infection in a cell-based assay to
Output: Identification of Novel Small-Molecule Inhibitors of Severe Acute Respiratory Syndrome-Associated Coronavirus by Chemical Genetics use of large collections of small molecules (chemical libraries) to investigate protein/chemical interactions