Figure 2. Tiered screening procedure utilized with the LOPAC¹²⁸⁰ library screen.

Compounds were passed through 4 tiers of screening for both IspC (teal) and IspD (purple). For IspC, these included a primary screen with YpIspC, a secondary screen with MtbIspC, a tertiary screen with MtbIspC coupled with FtIspD, and a quaternary antibacterial assay. For IspD, these included a primary screen with FtIspD, a secondary screen with EcIspD, a tertiary screen with pyrophosphatase, and a quaternary antibacterial screen. Cut-off values for each screen are indicated (% residual enzyme activity or % residual growth). Compounds retained through the quaternary screen for both IspC and IspD were considered lead compounds and were examined for mechanism of inhibition.