Imaging Assays to Detect DNA Damage in Trypanosome Parasites Using γH2A

Area of Science:

• Parasitology
• Molecular Biology
• Drug Discovery

Background:

• Neglected tropical diseases like Chagas disease and human African trypanosomiasis (HAT), caused by trypanosomatid parasites, represent a significant global health challenge.
• Current treatments for Chagas disease are outdated, lengthy, and associated with severe side effects, highlighting the urgent need for new therapeutic strategies.
• Cyanotriazole compounds have shown promise by selectively inhibiting trypanosome nuclear topoisomerase II, but their mechanism of parasite death requires further elucidation.

Purpose of the Study:

• To develop and validate robust assays for detecting DNA damage in trypanosome parasites.
• To confirm that the inhibition of topoisomerase II by cyanotriazoles leads to lethal double-strand DNA breaks.
• To establish high-throughput methods for evaluating potential anti-parasitic compounds targeting DNA damage pathways.

Main Methods:

• Development of immunofluorescence-based assays utilizing the γH2A marker to detect DNA damage in intracellular amastigotes of *Trypanosoma cruzi* and bloodstream-form *Trypanosoma brucei*.
• Implementation of robust DNA damage pathway-based cellular assays to assess the efficacy of topoisomerase II poisons.
• Establishment of a 384-well plate protocol for high-throughput screening of compounds causing DNA damage via γH2A measurement in *T. cruzi*.

Main Results:

• Successful development of immunofluorescence assays to detect the γH2A DNA damage response in both *T. brucei* and *T. cruzi* parasites.
• Demonstration that topoisomerase II inhibition in trypanosomes results in detectable DNA damage, specifically the phosphorylation of H2A (γH2A).
• A high-throughput 384-well plate assay for *T. cruzi* allows for efficient evaluation of compounds inducing DNA damage.

Conclusions:

• The developed γH2A immunofluorescence assays provide a reliable method for assessing DNA damage in trypanosome parasites.
• These assays confirm that topoisomerase II poisons induce lethal DNA breaks, validating this as a viable mechanism for anti-parasitic drug development.
• The high-throughput *T. cruzi* assay facilitates the discovery of novel anti-parasitic compounds for neglected tropical diseases, with potential for adaptation to other pathogens.