Identification of Small-Molecule Inhibitors of Nucleoside Triphosphate Hydrolase in Toxoplasma gondii

Approximately 150,000 small-molecule compounds were tested by a robotic screening assay for their ability to inhibit nucleoside triphosphate hydrolase (NTPase), a novel enzyme of the tachyzoite form of Toxoplasma gondii. Five unrelated species of compounds were found to inhibit the activities of both NTPase isoforms (NTPase isoform I [NTPase-I] and NTPase-II). The 50% inhibitory concentrations (IC(50)s) ranged from 0.1 to 20 μM, and in general, the IC(50)s were similar for both NTPase isoforms. However, the activity of NTPase-I was 20 times more sensitive than the activity of NTPase-II to one of the inhibitors: 9-hydroxy-10-(pentachlorophenoxy)stearic acid. The five compounds identified also prevented tachyzoite replication in vitro, with IC(50)s ranging from ∼7 to ≥50 μM. The most effective of these initial compounds, 2-phenylthio-indole, was used to identify six additional, structurally related compounds, which were tested for their inhibitory effects on enzyme activities and tachyzoite replication. Surprisingly, these compounds were competitive inhibitors of NTPase-I but noncompetitive inhibitors of NTPase-II. Modifications to the indole and phenol rings resulted in alterations of activity, thus providing insight into the structural features that are important for inhibition of T. gondii NTPases.