Subcellular Localization of Photoreactive Ethidium Analogs in Trypanosoma brucei by Fluorescence Microscopy

Abstract

To identify the in vivo targets of the trypanocide, ethidium bromide, the fluorescent staining of T. brucei was examined for a series of ethidium analogs using fluorescence microscopy. Determination of the biological targets for most drugs is limited by the reversible nature of their interactions. To overcome this limitation, photoaffinity (azido) analogs of ethidium, which are capable of covalent attachment with photoactivation, were used to identify the ethidium binding sites within the parasites. Two of these compounds, when covalently attached, demonstrated an enhancement of fluorescent staining and were selective for the kinetoplast at low drug concentrations. These compounds were also those found previously to have the highest trypanocidal activity. Propidium, a phenanthridinium analog identical to ethidium except for a larger, more ionic substitution at R5, showed more nonspecific binding as determined by its general staining of the cytoplasm.