Human foreskin fibroblasts (HFF) were purchased from ATCC, maintained in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal calf serum (FCS, Gibco-BRL, Waltham, MA, USA) and antibiotics as previously described . Transgenic T. gondii β-gal (expressing the βgalactosidase gene from Escherichia coli) were kindly provided by Prof. David Sibley (Washington University, St. Louis, MO, USA) and were maintained, isolated, and prepared for new infections as shown before . The screening cascade for the compounds was described in former studies 114 . All the compounds were prepared as 1 mM stock solutions from powder, in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). For in vitro activity and cytotoxicity assays, HFF were seeded at 5x10 3 /well and allowed to grow to confluence in phenol-red free culture medium at 37°C and 5% CO2. T. gondii tachyzoites were released from host cells, and HFF monolayers were infected with freshly isolated parasites (1x10 3 /well), and compounds were added concomitantly with infection. In the primary screening, HFF monolayers infected with T. gondii β-gal received 0.1 and 1 µM of each compound, or the corresponding concentration of DMSO (0.01 or 0.1% respectively) as controls and were incubated for 72 h at 37˚C/5% CO2 as previously described 126 . For the dose-response study (IC50 values), measurements for T. gondii β-gal were performed. The selected compounds were added concomitantly with infection in 8 serial concentrations 0.007, 0.01, 0.03, 0.06, 0.12, 0.25, 0.5, and 1 μM. To measure the activity of the tested compound on T. gondii β-gal parasites, after a period of 72 h of culture at 37°C/5% CO2, culture medium was aspirated, and cells were permeabilized by adding 90 μL PBS (phosphate buffered saline) with 0.05% Triton X-100. Next, chlorophenolred-β-D-galactopyranoside (CPRG; Roche Diagnostics, Rotkreuz, Switzerland), substrate for the β-galactosidase of 10 μL 5 mM chlorophenolred-β-D-galactopyranoside (CPRG; Roche Diagnostics, Rotkreuz, Switzerland) in PBS was added. Release of chlorophenol red was measured at 570 nm using an EnSpire® multimode plate reader (PerkinElmer, Inc, Waltham, MA, USA). A 100% proliferation of T. gondii β-gal was assigned to control parasites treated only with DMSO. For the primary screening at 0.1 and 1 μM, the activity was measured as the release of chlorophenol red over time and was calculated as percentage from the respective DMSO treated control, which represented 100% of T. gondii β-gal growth. For the IC50 assays, the activity measured as the release of chlorophenol red over time was proportional to the number of live parasites down to 50 per well as determined in pilot assays. IC50 values were calculated after the logit-log-transformation of relative growth and subsequent regression analysis. All calculations were performed using the corresponding software tool contained in the Excel software package (Microsoft, Redmond, WA, USA). Cytotoxicity assays using uninfected confluent HFF host cells were performed by the alamarBlue assay as previously reported 127 . Confluent HFF monolayers in 96 well-plates were exposed to 0.1, 1 and 2.5 μM of each compound. Non-treated HFF as well as DMSO controls (0.01%, 0.1% and 0.25%) were included. After 72 h of incubation at 37°C/5% CO2, the medium was removed, and the plates were washed once with PBS. Resazurin suspended in PBS to a final concentration of 0.01 g/L was added to each well (200 μL/well). Plates were measured at excitation wavelength 530 nm and emission wavelength 590 nM using an EnSpire ® multimode plate reader (PerkinElmer, Inc). The cytotoxicity values were calculated as percentage of the respective DMSO control, which represented 100% of HFF viability.