The importance of balancing potency vs. MRSA and cytotoxicity led us to examine a subset of compounds more thoroughly for toxicity against the human embryonic kidney cell line HEK293. We measure toxicity through either CC50 (concentration of compound inducing 50% reduction in viability) or Dmax as defined by CO-ADD and calculated following exposure of cells to compounds and recording of metabolic activity via either tetrazolium dye absorbance or resofurin fluorescence (methods are described in Supplementary Information -Biology). Dmax is a parameter which quantifies toxicity at the highest tested concentration (32 µg/mL for all compounds), calculated as reciprocal percentage of viable cells (100 -viability %) where cells were either allowed to recover or viability was measured immediately after treatment. While some studies suggest that 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide is less sensitive than resofurin, 20 others report overestimation of viability due to accumulation of resofurin in cells or the opposite due to extensive reduction of the dye by cells with high metabolic activity. Our efforts focused on trying to replicate the original toxicity experiments (by CO-ADD) with tamoxifen using a protocol modified for use with 96-well plates. Given the variability of protocols and results in the literature it was important to compare with a standard protocol we have reliably used in our own laboratory. When both CC50 values and Dmax are mapped against the other two experimental variables (i.e., recovery status after treatment (yes/no) and the detection output (MTT/resazurin)) the majority of compounds were found to be toxic as classified by CO-ADD with a small spread of the data for most variations of protocol used (Figure ). Truly "non-toxic" compounds might be expected to show Dmax < 50% for all experiment variations, e.g., 861. The Dmax spread for the control compound (tamoxifen) across all experiments was small and in agreement with previously-measured values (CC50 9±2 µg/mL). A small difference in Dmax irrespective of whether cells were allowed to recover suggests those compounds are cytostatic or induce a partial response through a mechanism of toxicity which results in slowed cell growth or cause cell death through mechanisms the cells can resist, for example compounds 821, 822, 869, 979, 982, 983 and 984. A large spread of Dmax values comparing recovery and nonrecovery conditions larger than 20% is perhaps indicative of a mechanism of toxicity that persists beyond exposure, i.e., cytotoxic compounds that continue to induce toxicity as cells recover and restart dividing, for example compounds 865, 870, 871, 980 and 981. CC50 values reflect these classifications (Figure ) with 861, 862 and 869 being least toxic compared to tamoxifen (CC50 5.3-15.5 µg/mL) while other compounds exhibit CC50 values associated with higher toxicity (1-6 µg/mL). Where resofurin fluorescence was the measured output, data are more variable (e.g., 862 was found to be non-toxic by both resazurin assay as well as with MTT after recovery, but toxicity more than 50% was measured in the acute exposure by MTT). This variability could be driven by interference of the compounds with the fluorescence readout or resofurin build-up as previously described. Another limitation to consider is the solubility of the compounds; while all were tested at concentrations ranging from 32 µg/mL (approximately 100 µM for a 300 Da compound) to 0.00625 µg/mL (~ 0.19 µM) with a dilution factor of 2, all compounds screened against HEK293 except for 821, 861 and 870 have aqueous solubility less than 100 µM. Consequently, compounds precipitating during the assay or unpredictable interactions with serum in the media (and as a result interaction with the cells and their transport in the cell) may explain the spread of the data shown, in particular for those with very low aqueous solubility, such as 983. An attempt to characterize the mechanism of toxicity led to testing compound interaction with DNA molecular beacons, which are short strands of DNA tagged with a donor-acceptor pair of fluorophores. The proximity between the fluorophores when the DNA strand is correctly folded results in no fluorescence from the donor; should a compound interfere with DNA folding, a change in melting temperature will be observed. No compounds tested showed reduction in DNA melting temperature (Figure Supplementary Material -Biology), meaning compounds did not interfere with DNA folding. By comparison, doxorubicin showed a dose-response behavior and is a known DNA intercalator.