4.2. Library Screening

The compounds screened in this work correspond to all 1200 off-patent FDA-approved drugs for human use in the Prestwick Chemical Library (Prestwick Chemical, Illkirch-Graffenstaden, France). Stocks were 10 mM in dimethyl sulfoxide (DMSO). All of them were first diluted 20-fold in sterile Milli-Q water, resulting in a drug array at 500 µM in 5% DMSO.

A pre-inoculum (12 mL) of S. pneumoniae R6CIB17 was grown until the early exponential phase (OD₅₅₀ of approximately 0.1) and then aliquots of 180 µL were disposed of in a 96-well plate using a fully automated pipette (Rainin E4 XLS+, Mettler Toledo, Columbus, OH, USA) (1.8 × 10⁷ CFU per well). Then, 20 µL of each diluted drug was added to obtain a final volume of 200 µL (final concentration: drug 50 µM in 0.5% DMSO). The plate was introduced in a Multiskan GO (Thermo Fisher Scientific), and the growth curve at 37 °C was monitored using OD_550. The blanks containing compounds without bacteria were subtracted since some of the compounds showed absorbance at 550 nm. The controls were run in parallel in every plate, containing only bacteria in culture media with and without DMSO 0.5%. All experiments were performed in duplicate.