We trained virtual screening models on new and previously generated datasets for the PriA-SSB target. This target is a protein-protein interaction that is involved in bacterial DNA replication restart and was considered as a candidate target for antibiotics development. The compounds in these datasets come from four batches of a Life Chemicals Inc. (LC) library designated LC1-LC4 and the Molecular Libraries Probe Production Centers Network (MLPCN) library (Table ). The compounds considered for prospective evaluation come from the in-stock AMS library and the synthesize-on-demand Enamine REAL library. 60 We screened the MLPCN compound library using an AlphaScreen (AS) assay at a single concentration (33.3 µm) following the same protocol previously used to screen the LC1234 libraries. Briefly, library compounds and controls were dispensed from 10 mM DMSO stocks into white 1,536-well plates with an Echo 550 acoustic liquid handler. Next, 3 µL of a master mix was added to each well using a Mantis liquid handler with a high-volume silicone chip to yield a final reaction mixture containing 10 mM HEPES-HCl (pH 7. Plates were centrifuged briefly and rocked for an hour at room temperature. Then, the AS signal of each well was measured with a PheraStar plate reader using a 0.1 s settling time, 0.3 s excitation, a 0.04 s delay, and a 0.6 s integration time. AS signals for each well were adjusted as previously described to reduce the impact of plate and edge effects. We calculated a Z' factor for each plate and repeated plates with Z' < 0.5. In addition, we calculated the % inhibition relative to the controls for each compound. We retested compounds with ≥ 35% inhibition that also passed PAINS filters with the same assay to confirm activity.