The TIRAP-GyrB, MyD88-GyrB, and TRAF6-GyrB cell lines were stored in multiple aliquots as frozen stocks and thawed for HTS in a standardized manner 10 days (five passages) before HTS. Phenol red–containing standard DMEM was replaced by growth medium without phenol red during HTS assays. The concentration of FBS was not optimized toward lower concentrations because all follow-up experiments had been established at 10%. In the primary HTS, TIRAP-GyrB cells (10,000 cells in 25 μl of assay medium) were seeded in white 384-well solid-bottom tissue culture–treated plates (PerkinElmer) with a WellMate dispenser (Thermo Scientific Matrix), and plates were incubated in an automated tissue culture incubator (LiCONiC Instruments). After 24 hours, DMSO stock solutions of test chemicals (the St. Jude Bioactive collection), along with coumermycin A1 (Sigma-Aldrich), PS-1145 (Sigma-Aldrich), staurosporine (LC Laboratories), or DMSO (Thermo Fisher Scientific), were transferred with a Pin Tool (V&P Scientific) equipped with a 10H pin head to give a final test chemical concentration of 12 μM, along with 100 nM coumermycin A1 (one replicate per compound). In addition, groups of wells with 100 nM coumermycin A1 alone and 100 nM coumermycin A1 with 30 μM PS-1145 or 40 μM staurosporine were included in each plate as well. The final DMSO concentration in each well was 0.24%. Twenty-four hours later, each plate was treated with 5 μl per well Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific)–diluted (1:12 dilution) AlamarBlue (Invitrogen), incubated at 37°C for 1 hour, cooled down to room temperature for 15 min, followed by fluorescence analysis using an Envision HTS microplate reader (PerkinElmer). Next, the luminescence signals were measured using SteadyLite HTS luminescence assay reagent (PerkinElmer) and the Envision plate reader. In the AlamarBlue cell toxicity assay, the 40 μM staurosporine with 100 nM coumermycin A1 group and the 100 nM coumermycin A1 alone group were assigned as the positive (100% inhibition) and negative (0% inhibition) controls, respectively. In the luminescence assay, the 30 μM PS-1145 with 100 nM coumermycin A1 group and the 100 nM coumermycin A1 alone group were assigned as the corresponding positive (100% inhibition) and negative (0% inhibition) controls. Test compound activity was normalized to those of positive and negative controls in the individual assays. Two hundred thirteen unique chemicals with luminescence inhibitory activity ≥50% and with AlamarBlue cytotoxic inhibitory activity ≤20% were selected for further dose-response confirmation testing.

In the dose confirmation tests, the basic primary screening protocol was followed with minor modifications, including that 10 concentrations from 56 μM to 2.8 nM, along with 100 nM coumermycin A1 in triplicates, were tested. The activity data for individual chemicals were fit into sigmoidal dose-response curves if applicable to derive IC50 values with GraphPad Prism 7.00 (GraphPad Software).