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Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library

Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL

Nice Shindo

Mindy Sosa

High Throughput Screening Center, Southern Research Institute, Birmingham, AL

Thomas Fletcher, III

Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL

E. Lucile White

High Throughput Screening Center, Southern Research Institute, Birmingham, AL

Subramaniam Ananthan

Department of Chemistry, Southern Research Institute, Birmingham, AL

Colleen B. Jonsson

Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, AL

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV–induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro—compounds which will serve as excellent lead candidates for further evaluation. At a 10-µM concentration, 3 compounds with selective indexes (SI₅₀) of > 53 were discovered.

Key Words: high-throughput screening • severe acute respiratory syndrome • coronavirus • cytopathic effect

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