Fig. 2.

Spiroindolones rapidly diminish protein synthesis in the parasite. The rate of parasite protein synthesis was evaluated by monitoring [³⁵S]-radiolabelled methionine and cysteine ([³⁵S]-Met/Cys) incorporation into asynchronous cultures. Parasites were assayed for 1 hour in the presence of NITD609 (inverted triangle), anisomycin (diamond), cycloheximide (square), artemisinin (circle), or mefloquine (triangle), then extracted for radiographic measurements. Radiolabel incorporation was measured against inhibitor dosed over a five-log concentration range and percent incorporation was calculated by comparison to cultures assayed in the absence of inhibitor. Anisomycin and cycloheximide were included as positive controls. (A) Spiroindolone treatment rapidly diminishes protein synthesis in Dd2; however, this effect is mostly absent in (B) NITD609-R^Dd2 clone #2 except at very high concentration. 50% inhibition of [³⁵S]-Met/Cys incorporation was observed with 3x and 78x IC₅₀ of NITD609 on the NITD609-treated Dd2 wild type and NITD609-R^Dd2 drug-resistant clones respectively. Data are expressed in mean±SD and represent three independent experiments performed in triplicate. Similar losses of protein synthesis inhibition upon NITD609 treatment were observed in the resistant clones NITD609-R^Dd2 #1 and #3 respectively (see Fig. S2).