Photoaffinity labeling is a powerful stragegy for the study of drug action because it provides a means to identify, directly and unambiguously, drug targets within intact cells. This is especially pertinent to chemotherapeutic agents such as antiparasitic, anticancer, and antibiotic drugs since the contrasts in drug fates between the host and the target cells, and between sensitive and resistant cell lines, are important clues to the mechanisms of action. This project will identify the sites of drug binding which kill Trypanosoma brucei brucei (African Sleeping Sickness). Specific photosensitive analogs have been prepared from the trypanocidal drugs, ethidium and acriflavine, which appear to be authentic photoaffinity probes. These have been used to identify high affinity sites within trypanosomes through photolytically-induced covalent attachment shown by fluorescence of the intact parasite and by cell fractionation. The identity of the sites accounting for drug action, however, is unknown. Although we confirmed a selectivity for the kinetoplast, we have also shown that a dyskinetoplastic organism is not resistant. Recently, we have produced specific antibodies against the covalent drug adducts from photoaffinity labeling with the ethidium probe with which we can visualize adducts in parasites photolyzed with as little as 10-7 to 10-8M drug. We have also identified parasite strain differences in drug sensitivity and a tenfold synergism between the photoaffinity probe and ethidium. Photoaffinity labeling will be used in conjunction with antibody detection to localize drug sites within parasites using immunocytochemistry, both at the ultrastructural and the light and fluorescent microscopic level, and by cell fractionation. Parasite strain differences in response will be correlated with drug targeting. Strains already under study show a tenfold difference in drug response, and additional resistant strains will be selected. The synergism between ethidium and trypanocidal effects of photoaffinity labeling and the competition with active and non-active analogs will be explored for induced differences in drug targeting. The specific antibodies represent a substantial improvement in sensitivity for drug adduct detection, and should permit the definition of the drug targets at relevant drug concentrations. This is, therefore, a continued systematic study to determine the mechanisms of antiparasitic drug action through defining the intracellular drug disposition correlated with biologic action. These studies provide a general strategy for the use of photoaffinity labeling to study drug action.
| 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 |
