Delivering small molecule chemical series that give full cure in the Chagas disease mouse model of infection is proving challenging, despite compounds having high potency against the parasite and good oral pharmacokinetics. It is therefore proving important to try and identify where remaining parasites are residing directly following ending of drug pressure in order to identify those tissues/organs compounds need further optimisation for to deliver therapeutic levels.
In order to identify where the parasites are residing, histopathology methods will be optimised for whole tissue and whole organ clearing and immunolabelling. The aim is to develop clearing protocols for organs/tissues including; the brain and liver, spleen, heart, lymph nodes, ovaries, visceral fat, lungs, skeletal muscle, stomach and small and large intestines. The protocols will be based on the iDISCO method which has proved to be the most robust and quickest. This approach will be used on control and infected animals (BALB/c mice). Organs and tissues of healthy animals will serve as a control, while infected animals will be subjected to immunolabelling which will stain the parasites. The SSP4 monoclonal antibody is currently thought to be the best candidate for immunolabelling of T. cruzi. It targets a major surface glycoprotein SSP4 that is bound to the membrane via a GPI anchor characteristic to the amastigote form of T. cruzi parasites. Further work will also investigate whether the bleaching step is necessary for the protocol. We will also explore whether simply using dual fluorescent /bioluminescence T. cruzi (either CL Brener Luciferase/mNeon or another T.cruzi strain + Td tomato), it is possible to see the parasites in cleared tissues with fluorescence alone and avoid the need for more expensive immunolabelling of remaining parasites. Additionally, a less invasive non-methanol protocol (uDISCO) will also be carried out on multiple organs to compare the results from both methods and see what offers the better option.
Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI MSI) has become a very powerful tool for studying the localization of endogenous and exogenous compounds, in particular drug distribution in biological tissues. Knowledge of the deposition and distribution of drug compounds and their metabolites is essential in drug discovery and development as it provides important information to the understanding of the efficacy, toxicology and pharmacokinetics of the drug. We intend to use mass spectrometry imaging, predominantly MALDI MS imaging, and associated histopathological techniques to understand:
- Compound distributional properties in those organs and tissues where remaining parasites are identified following drug treatment and,
- To attempt to identify and visualize parasite-specific biological markers, e.g. lipid or small peptide markers, and thus directly provide molecular information on the parasite distributions within the host(s), complementary to high-spatial resolution staining and pathology approaches. The analysis of an inactivated, in-vitro culture of the parasite could be a primary source of sufficient material to identify unique, specific lipid and peptides. The specific biological markers will then be used for the detection of parasites in infected animals
This project is supported by an MRC iCASE PhD in partnership with GlaxoSmithKline with WCAIR support for the animal disease model.