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High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses
Authors:Bara Cihlova  Andrea Huskova  Jiri Bserle  Radim Nencka  Evzen Boura  Jan Silhan
Institution:Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo namesti 2, 166 10 Prague 6, Czech Republic; (B.C.); (A.H.); (J.B.); (R.N.); (E.B.)
Abstract:Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.
Keywords:high-throughput screening  virus  drug  discovery  papain-like  protease  SARS-CoV-2  flavivirus  TBEV
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