Identification of small-molecule inhibitors of nucleoside triphosphate hydrolase in Toxoplasma gondii

Abstract

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 microM, 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 approximately 7 to > or =50 microM. 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.

FIG. 1.

Chemical structures of five species of compounds that where initially identified on the basis of inhibition of NTPase-II activity. The compounds are 2-{2-[2-(2,4,5-trichlorophenoxy)ethoxy]ethoxy}acetic acid (compound 1), 9-hydroxy-10-(pentachlorophenoxy)stearic acid (compound 2), urosolic acid (compound 3), 1H-1,2,3-triazole-4-carboxamide, 5-amino-1-({3,5-dichloro-4-[tricyclo(3.3.1.13,7)dec-1-ylcarbony]phenyl}methyl) (compound 4), and 2-phenylthio-indole (compound 5).

FIG. 2.

Chemical structures of 2-phenylthio-indole and derivatives. The compounds are 2-phenylthio-indole (compound 5), 2-[(3-nitrophenyl)thio]-1H-indole (compound 6), 2-(2-naphthalenylthio)-1H-indole (compound 7), 2-[(4-methoxyphenyl)thio]-1H-indole (compound 8), 3-phenylsulfenyl indole (compound 9), 2-(1-naphthalenylthio)-1H-indole (compound 10), 2-(4-nitrophenylthio) indole (compound 11), and 3-metylthio-2-(4-nitrophenylthio) indole (compound 12)

FIG. 3.

Double-reciprocal Lineweaver-Burk plots of the activities of NTPase-I (A) and NTPase-II (B). Activities were determined with various substrate concentrations in the absence (none) or presence (5 μM compound 5) of the inhibitor. The inhibition profile for NTPase-I activity is consistent with a noncompetitive mechanism, while the inhibition profile for NTPase-I activity is consistent with a competitive mechanism.