Variomag

The solubility of Prx was measured as a function of ethanol concentration up to 20%. Briefly, an excess amount of Prx was accurately weighed and placed into the glass screwed cap bottle. The appropriate volume of ethanol and water was added to obtain varying percentages of ethanol: 1%, 3%, 5%, 7%, 10% and 20% by volume respectively. The bottles were sonicated using ultrasonic cleaner (Elma, Singen, Germany) of 50 kHz for a few minutes to ensure homogeneity of the mixtures and then placed on the magnetic plate (Variomag^®, Thermo Fisher Scientific, Waltham, MA, USA). The mixture was stirred using a small magnetic bar at 150 rpm for 48 h in a thermostatic incubator with temperature controlled at 25 ± 0.1 °C. The mixture was then passed through a 0.45 μm membrane filter to obtain a clear filtrate. The filtrate was diluted if necessary and the absorbance was measured at 359 nm using a UV-spectrophotometer (UV-2450). The absorbance was converted into Prx concentrations using the calibration curve. The influence of ethanol on Prx solubility was assessed by plotting the concentration of Prx against the percentage of ethanol. The results were obtained using Yalkowsky and Roseman’s logarithmic—linear model [24 ].

The dose-response validation was done in an extended set of cell-based luciferase reporter assays. Fresh compound solutions of those active in the primary screens were retrieved from the frozen library and diluted in DMSO to ECHO-qualified 384-well cyclic olefin copolymer plates (Labcyte). On the day of the experiment, reporter cells were harvested and resuspended into phenol red-free DMEM culture media at a concentration of 500 cells/μl. Four-microliters aliquots of the cell suspension were dispensed to white polystyrene 1536-well plates (Corning) by a Multidrop Combi liquid dispenser (Thermo Scientific). The test compounds were transferred from the 384-well compound plates to 1536-well assay plates using contact-free acoustic transfer by ECHO 520 (Labcyte) integrated in the fully automated robotic HTS station Cell::Explorer (Perkin Elmer). The compounds were tested in triplicates at eight different concentrations in the range from 10 μM to 5 nM. Each plate was shaken for 30 s using the plate shaker Variomag (Thermo Scientific) and then incubated at 37°C and 5% CO2 in humidified atmosphere for 24 h.

Experiments were performed as previously described by Wayne et al. Briefly, screw-capped glass tubes (Max Wiegand, Germany, total volume: 18.5 mL) have been filled with 10 mL mycobacterial culture (OD = 0.004; Head Space Ratio (HSR): 0.54), tightly closed, wrapped with Parafilm and incubated at 37 °C on a magnetic stirring plate (Variomag, Thermo Scientific) at 130 rpms 7–35 days. During oxygen starvation, OD₅₉₄ was measured with a portable photometer (pHotoFlex STD, WTW) without opening the tubes. Oxygen consumption was monitored via decolorization of the redox indicator methylene blue (1.5 µg/mL) in control tubes. Methylene blue served as a visual indicator of hypoxia. Loss of blue color occurs after 10–17 days, depending on the strain. For aerated controls, loosely capped glass tubes with magnetic stirrer or square bottles were filled with 7 mL culture and incubated at 37 °C. For reactivation experiment, dormant cultures (35 days of oxygen starvation) were inoculated in fresh Dubos medium (supplemented with 10% OADC and 0.05% Tween 80) at indicated time points and incubated in aerated conditions at 37 °C.