# TRYPANOSOMES:  PHOTOAFFINITY LABELING OF ETHIDIUM SITE

> **NIH NIH R01** · UNIVERSITY OF TEXAS MED BR GALVESTON · 1988 · $119,123

## Abstract

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.

## Key facts

- **NIH application ID:** 3127399
- **Project number:** 5R01AI017700-09
- **Recipient organization:** UNIVERSITY OF TEXAS MED BR GALVESTON
- **SAM.gov UEI:** MSPWVMXXMN76
- **Principal Investigator:** K L YIELDING
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1988
- **Award amount:** $119,123
- **Award type:** 5
- **Project period:** 1987-09-01 → 1990-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3127399

## Citation

> US National Institutes of Health, RePORTER application 3127399, TRYPANOSOMES:  PHOTOAFFINITY LABELING OF ETHIDIUM SITE (5R01AI017700-09). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3127399. Licensed CC0.

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