Eleven amino-substituted 1,4-naphthoquinones were synthesized via the reaction of 1,4-naphthoquinone with different primary and secondary mono-and diamines in the presence of dichloromethane ethanol (1:2) solvent at room temperature. All compounds were purified by flash column chromatography, characterized by TLC, HPLC, 13 C-NMR, 1 H-NMR, and FT-IR spectral analysis and were evaluated in vitro for antifilarial activity using adult bovine filarial worm Setaria digitata by assessing worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction. Seven of the 11 compounds had macrofilaricidal activity with compounds 9 (2-[(1,3-dimethylbutyl) amino] naphthalene-1,4-dione) and 11 (2-(4-methylpiperazin-1-yl) naphthalene-1,4-dione) having maximum activity (ED 50 values of 0.91 and 1.2 mM, respectively, at 48 h). The effect of different substitutions on antifilarial activity is discussed. Drug Dev Res 71: 188-196, 2010.

Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature.

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 +/- 3.41 mg ml(-1), 0.09 +/- 0.019 micromol min(-1) ml(-1), and 0.009 +/- 0.002 micromol min(-1) mg(-1) protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC(50) values were 19.42, 51.41, and 114.86 microM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 microM, respectively, at 48-h incubation while curcumin was effective at 54.29 microM. In MTT reduction assay, the ED(50) values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 microM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.