This project seeks to understand the mechanisms of action for antiparasitic drugs through identification and study of drug binding sites within the infective parasite. Specifically, photoaffinity labeling will be used to identify drug receptors for ethidium, and related drugs, in Trypanosomes. Drug-receptor adducts will be identified by quantitative fluorescence microscopy, electron and light microscopic autoradiography, and by biochemical separation studies. We have established that azido analogs of ethidium prepare in our laboratories are authentic probes for ethidium binding; and that the covalent adducts produced supravitally in Trypanosoma brucei by photoactivating the probes in situ are highly relevant to antiparasitic action. Our fluorescence studies have demonstrated preferential pulse photoaffinity labeling of kinetoplasts and correlated trypanocidal action using probe concentrations as low as 107M. This putative target will be subjected to intensive studies to determine: can labeling be blocked (or intensified) by the parent ethidium or other active and inactive drugs; can labeling be produced by ineffective analogs; do drug resistant parasites show identical or different patterns of labeling; and can the remaining kinetoplast DNA or other kinetoplast constituents be labeled in dyskinetoplastic strains of parasites. In all instances, drug labeling studies will be correlated with measurements of antitrypanosomal activity. After labeling of the intact parasite, kinetoplast DNA will be isolated immediately and at subsequent intervals to follow the localization and fate of the bound drug. This will be done by double labeling and by restriction mapping. Furthermore, the nature of the changes produced in the DNA with drug-evoked dyskinetoplasia will be examined by restriction analysis. These studies will attempt to establish the essential nature of the kinetoplast as a drug target in comparison with other drug targets. The precise time course of the development of ultrastructural changes and the accompanying changes in synthetic functions for DNA, RNA, protein and membrane components will be determined following pulse photoaffinity labeling. A concerted effort will be made to establish a cell culture of T. brucei to enhance these investigative efforts. Success in these experiments should provide major progress in understanding and designing drug therapy for parasitic diseases.
| Omholt, P E; Cox, B A; Prine, L C et al. (1993) Use of drug-specific antibodies to identify ethidium adducts produced in Trypanosoma brucei by photoaffinity labeling. Acta Trop 55:191-204 |
