Chagas disease is a chronic infection caused by the parasite Trypanosoma cruzi (T. cruzi) and is transmitted to mammals by hematophagous triatominae insects, often known as “kissing bugs”. The development of the parasite in the heart muscle or digestive tract causes severe damage, resulting in organ failure. The current treatments, benznidazole and nifurtimox, have a limited efficacy, show heavy side effects, and developing resistance. Increasing evidence shows that epigenetic targets play a crucial role in the parasitic life cycle of protozoan parasites.1 Our research focuses on post-translational modifications (PTMs) of histone tails, especially acetylation. Bromodomains (BDs) are readers of the acetylated lysine residues. The bromodomains of the T. cruzi parasite have been briefly described in the literature, but their function remains uncharacterised. The life cycle of the parasite is complex and is thought to rely heavily on epigenetic control, hence our main interest is to validate the T. cruzi bromodomains as therapeutic targets. Optimisation of expression and purification protocols of the bromodomain containing factors (BDFs) protein constructs in E. coli was followed by the development of a waterLOGSY-based assay to screen acetyl-lysine mimic containing compounds. The binding of the hits was further characterised by 1H ligand-observed protein titration to obtain KD, as well as by 19F NMR. A known human bromodomain ligand was characterised to have a KD value between 25 and 55 µM, and is the current highest affinity ligand for TcBDF3 identified by 1H NMR. A focused library of analogues was synthesised to validate the binding of the chosen acetyl-lysine mimicking motif, then several probes were designed to develop new assays for T. cruzi bromodomains. Firstly, diazirine-derived probes were synthesised to produce photo-activatable covalent binders that can be monitored by protein mass spectrometry. Additionally, four fluorescent probes, with varying linkers, were synthesised to establish a fluorescent polarisation assay. In summary, photo-activatable diazirines, and fluorescent probes were synthesised to provide tool compounds for further assay development to study the T. cruzi bromodomains. Identified ligands will show validation of bromodomains of T. cruzi as targets for Chagas disease. This will have wide implications in the use epigenetics targets to treat parasites more broadly.

Chemical tools to probe the role of bromodomains in the parasite Trypanosoma cruzi

2019 Spring National Meeting

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