Fig 5. Screening cascade for DOS screen (A) and potency comparison of hits from the DOS library between the novel axenic and intramacrophage assay (B).

A: Screening cascade. Numbers of compounds that passed and failed the hit selection criteria are shown respectively in green and red. Numbers marked with an asterisk indicate that the selectivity window is unknown and may be ≥ 10 (no effect seen in HepG2 assay, activity in the cidal axenic assay pEC50<5.3). The cascade starts with screening 9,907 compounds in the novel axenic assay at a single concentration (nov. axenic SP). Identified hits are processed in potency format in both the novel axenic assay (axenic pot., for activity confirmation) and HepG2 assay (HepG2 pot., toxicity information against the human cell line HepG2). Active compounds that provide a ≥ 10-fold toxicity window are processed in the intramacrophage potency assay (intramac. pot.) for hit confirmation, resulting in 24 hits. B: Comparison of novel axenic and intramacrophage mean pEC₅₀ values for 141 compounds. The size of the markers is inversely related to the toxicity against the THP-1 cells (i.e. high toxicity–small symbol). Colour is by series (green: Ortho Azetidine Nitrile, blue: Azetidine Nitrile, orange: Povarov, red: SnAr 8-ortho, yellow: other). Data set represents three biological replicates for the novel axenic assay (with the exception of one compound with two replicates only) and at least two replicates for the intramacrophage assay.