Thermal shift assays.

The thermal stability of recombinant CDPK1 in the presence or absence of BKIs was determined as previously described (35). Each assay well contained a reaction mixture of recombinant CDPK1 enzyme (4.4 μM), 20 μM inhibitor, and 5% dimethyl sulfoxide (DMSO) in TSA buffer (5% glycerol, 25 mM HEPES, 500 mM NaCl, 0.025% NaN₃, 2 mM dithiothreitol [DTT] with the pH adjusted to 7.25, with or without CaCl₂ or EGTA, as indicated). A negative control lacking inhibitor was present on each assay plate. Melting was measured using SYPRO Orange (at a final dilution of 4.5× from the 5,000× stock; Invitrogen), which fluoresces when it binds to exposed hydrophobic regions of the protein. All assays were performed in duplicate (or triplicate) independently at least two times. Preliminary experiments established that DMSO concentrations from 0 to 5% did not alter the T_ms of purified recombinant proteins (not shown).

L-CETSAs (20) were performed using the previously described modifications (24). Briefly, a mixture of T. gondii cells that had spontaneously egressed and those from the same culture that were mechanically released from host fibroblasts was washed in phosphate-buffered saline and the cells were counted. Parasites were resuspended in CETSA buffer (25 mM Tris HCl, pH 7.4, 2 mM DTT, 10 mM MgCl₂, 1 mM CaCl₂) plus phosphatase and protease inhibitor cocktail (Halt protease phosphatase inhibitor; Thermo Scientific) at 5 × 10⁸/ml. After three freeze-thaw cycles, lysates were clarified by centrifugation at 16,100 × g for 25 min at 4°C. Aliquots were incubated with 2 μM BKI (0.5% DMSO) for 10 min at 37°C. Thermal stabilization of CDPK1 was tested at 7 temperature points (66°C, 62°C, 58°C, 54°C, 50°C, 46°C, and 42°C in duplicate per compound at 2 μM) in a reaction volume of 20 μl with 10⁷ cell equivalents. Samples were heated for 3 min at the desired temperatures in the thermocycler and again centrifuged as described above to remove aggregated proteins. Taking care not to disturb the pellet, supernatants were removed and 2 × 10⁶ cell equivalents (∼4 μl) mixed with sample buffer was analyzed by SDS-PAGE, with the entire duplicate series for each compound in the experiment being run on a single gel. The gels were blotted and incubated with anti-TgCDPK1 (1:50,000) (24) plus goat anti-rabbit IgG IRDye 800 and imaged on a LI-COR Odyssey instrument. The blots were stained with Ponceau to detect potential artifacts, such as nonspecific protein precipitation or gel bubbles. Each compound was tested in at least two experiments, and the solvent control was replicated three times to yield an average value used for the calculations, as described previously (24). For repeat experiments, temperatures outside the range of interest were typically not included.

For IC-CETSAs, we followed the procedure described for mammalian cells (20, 36). Briefly, mechanically harvested parasites were pelleted and rinsed in Dulbecco modified Eagle medium (DMEM) (lacking serum) and then resuspended at 4 × 10⁸/ml in DMEM (which contains 1 mM CaCl₂) prewarmed to 37°C. Compounds were added in duplicate to yield a final concentration of 2 μM BKI and 0.5% DMSO. After thorough mixing, samples were incubated for 1 h at 37°C in a CO₂ incubator. The treated cells were centrifuged, rinsed in phosphate-buffered saline, and resuspended in TSA buffer containing protease and phosphatase inhibitors, as described above. Aliquots (25 μl) containing 10⁷ cells were then heated for 3 min at the desired temperatures and placed on ice until the range of heat treatments was complete. They were then subjected to three freeze-thaw cycles, and the clarified lysate was prepared and analyzed by Western blotting as described above. Preliminary experiments tested whether parasites remained viable during the 1-h incubation in the presence or absence of drug via microscopic analysis following staining with ethidium homodimer (0.5 μM) (not shown). The viability of parasites incubated with the BKIs used in this study remained above 94%. For IC-CETSAs, each compound was tested in at least two experiments, and the solvent control was tested in six experiments. The T_ms for each condition (compound and cell line) were averaged and used for the calculation of the thermal shift from the solvent control.