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- Title
Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.
- Authors
Sykes, Melissa Louise; Hilko, David Hugh; Kung, Livia Isabella; Poulsen, Sally-Ann; Avery, Vicky Marie
- Abstract
Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 μM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi. Author summary: Chagas disease occurs within 21 countries in the Americas, causes over 10, 000 deaths per year and a further 25 million people are at risk of being infected. The cause of Chagas disease is Trypanosoma cruzi, a single celled protozoan parasite, which enters the bloodstream of a host by the bite of a "kissing bug". In advanced disease stages, the parasite hides in heart and gut tissue and is difficult to treat. Identifying the replicative ability of these parasites is important to understanding Chagas disease progression and the effectiveness of compounds and drugs for treatment. By testing a panel of nucleoside analogues that may incorporate into DNA during synthesis, we developed an image-based method with a fluorescently-labelled DNA probe to identify replicating parasites. This method has effectively shown that drugs used to treat the parasite are able to clear intracellular infection, whilst a compound that was not efficacious in clinical trials leaves replicating T. cruzi behind. This methodology can be used to understand the action of further compounds and supports the identification of new, less toxic probes to assess intracellular parasite replication.
- Subjects
TRYPANOSOMA cruzi; INTRACELLULAR pathogens; ANTIPARASITIC agents; PYRIMIDINE nucleosides; PYRIMIDINES; DNA synthesis; CONENOSES; CHAGAS' disease
- Publication
PLoS Neglected Tropical Diseases, 2020, Vol 14, Issue 3, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0008068