biostudies-literatureSaragliadis ANovo Nordisk Fonden100025https://www.ebi.ac.uk/biostudies/studies/S-EPMC7388967biostudies-literatureUnknown5We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from <i>Yersinia enterocolitica</i> that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to cCell surface (Amsterdam, Netherlands)Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen.PMC7388967NNF18OC0032818Saragliadis ALinke DfalseAssay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen.We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from <i>Yersinia enterocolitica</i> that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to c2019-01-01T00:00:00Z2019 Dec2025-04-05T15:21:00.584Z2025-04-05T15:21:00.584ZS-EPMC73889673274314110.1016/j.tcsw.2019.100025