Fig. 1

The compound screening procedure. (a) Following assay development, stability and quality testing, the screening of the PCL against HAdV infection was performed. Imaging, image analysis and data processing were independently carried out at UZH and EPFL, before hit ranking. (b) Schematic overview of the wet-lab pipeline. PCL compounds and DFT positive control in DMSO as well as DMSO alone as negative control were pre-spotted onto 384-well imaging plates by Echo acoustic liquid handling at 10 nl corresponding to a final concentration of 1.25 µM in 80 µl assay volume/well and stored at −20 °C. Compound-blinded plates are thawed and 4,000 A549 cells/wells seeded. The following day, the cells were inoculated with HAdV-C2-dE3B at 1.77*10⁵ genome equivalents/well. Allowing for multiple viral replication rounds, the cells were PFA-fixed at 72 hpi and the nuclei stained with Hoechst 33342. The infection phenotypes were imaged using an epifluorescence HT microscope and scored using Plaque2.0. The data of the four technical replicates were further processed in R or through EPFL-BSF LIMS. (c) Exemplary epifluorescence microscopy images of cells in 384-wells stitched to a screening plate overview of 32 replicates of negative (two most left columns) and positive control (two most right columns) and 320 blinded PCL compounds (centre 20 columns). Hoechst-stained nuclei are shown in blue, viral GFP in green. (d) Representative 384-well epifluorescence microscopy images of the DMSO negative control (most left), the DFT positive control (most right) and the top hit Nelfinavir mesylate (centre). Empty black triangle indicates a plaque (infection focus) from a productively infected cell. White arrows point out infected cells that did not form a plaque. Hoechst-stained nuclei are shown in blue, infected cells expressing GFP in green. Scale bar is 5 mm.