Figure 3. Toxicity filtering of killing hits highlights semapimod.

A) Schematic of toxicity filtering steps. B) Relative viability of HepG2 and HEK293 in the presence of ~10 μM compound was determined using a resazurin-based cytotoxicity assay. Shown is the mean of two biological replicates. The shaded area indicates a relative viability of <0.5 in either cell line. C) Similarity of compounds with >0.5 HepG2 and HEK293 relative viability to known antibiotics and antiseptics. Compounds are separated by their curation method (primary screen or ML). Asterisks denote p < 0.05 with two-sided Mann-Whitney U test. D) High-throughput determination of metabolism dependence for the 49 selective stationary-phase killing compounds. (top) Killing efficacy in low or high metabolic conditions against Ec BW at 50 μM. Log survival was calculated by dividing the CFU/mL after antibiotic treatment by the initial CFU/mL to obtain percent survival, and taking the log-transformed value. (bottom) Metabolism dependence was estimated by dividing the change in cellular survival between high and low metabolic conditions by the change in bacterial intracellular ATP under these conditions¹⁶, and taking the negative of the resultant value. For comparison, data from known antibiotics are shown. Data are representative of two biological replicates. Negative x-axis values indicate less metabolism dependence. E) Selectivity index was calculated by dividing the IC₅₀ of HepG2 relative viability by the IC₅₀ of Ec BW stationary-phase killing. Dose-response curves were performed in biological duplicate. A summary of the IC₅₀ values can be found in Table S1. F) Hemolysis of rat Sprague-Dawley red blood cells was tested at 100 μM compound and normalized to the positive control (1% Triton-X 100). Data are representative of two biological replicates; error bars indicate s.e.m. G) Structure of semapimod.