K-070. Killing Effect of 3-Methyleneoxindol (MOI) and Glutathionyl Methyleneoxindol (GS-MOI) on Trypanosoma brucei YTAT1.1 Procyclic Forms in Vitro
Trypanosoma brucei , a protozoan parasite transmitted by the tsetse fly, is the etiologic agent of African trypanosomiasis which threatens millions of people and their domesticates in sub-Saharan Africa. Trypanothinone reductase (TR) is part of the enzymatic mechanism for removal of metabolically-generated reactive oxygen intermediates (ROIs) and inhibition of this enzyme results in parasite death. We are investigating the activity of two indole acetic acid derivatives, 3-methyleneoxindole (MOI) and glutathionyl MOI (GS-MOI), on procyclic trypanosomes in vitro. At concentrations as low as 10μM there is 90% killing of procyclic trypanosomes by one hour and inhibition of TR activity in crude homogenates. Enzyme assays, screening for the reduction of β-NADPH, were performed on a Biotek plate reader. Further purification of the crude cell homogenate by DEAE affinity chromatography, showed an enrichment for the enzyme in the 0.3M salt fractions. Initial assays in this purified homogenate show cleaner inhibition curves, and initial inhibition assays suggest similar levels of efficacy of MOI and GS-MOI as inhibitors. In order to assess potential cytotoxic effects on the host, viability assays were also performed in vitro on insect cell cultures, and on rat liver Clone 9 cells. These results suggest that neither molecule is cytocidal for these cell lines at the doses used to kill procyclics, increasing their potential as possible lead compounds in drug discovery efforts.