Figure 3. High-throughput screening of the compound library.

(A) (i) Flow diagram of the compound screen. Over 5000 compounds were dispensed into 24 of 384-well plates, enzyme was pre-incubated with compound before the addition of substrate and fluorescent intensity was read at an excitation 330 nm and emission at 420 nm with a Tecan Spark plate reader. (ii) Organization in 384-well plate. (B) Results of the screen. Two concentrations of the compound library were aliquoted- 1.25 µM and 3.75 µM. A single dot from the scatter plot represents each of the over 5000 compounds. There are 8 compounds and 29 compounds, in low and high concentration, respectively that reduce the PLpro activity more than 25%. Amongst those hits, four compounds were overlapping. In the low concentration (1.25 µM), one compound is auto-fluorescent and three have a Z score of more than −3.5.