Phenotypic screening enabled the discovery of drugs against Human African Trypanosomiasis (HAT), but questions about targeted proteins, mechanisms of action, and side effects remain unanswered. To address this, scientists developed methods for identifying cellular targets and mechanisms of lead compounds. Several successful target identification techniques exist, though some have drawbacks like proteome loss or strong background signals.

This project aims to overcome such limitations by creating and validating a novel system that combines SNAP-tag and BioID technologies based on the mutated biotin ligase BirA* protein. This fusion protein expressed within the parasite will facilitate the capture of small molecule targets in the cytosol and organelles of the Trypanosoma brucei (T. brucei).

Challenges were addressed, including designing a plasmid for homologous recombination and expressing a polycistronic gene in T. brucei. Different stable cell lines with integrated genes were obtained. Various tools were generated by modifying the BirA* protein or its linker within the fusion protein. The cMyc_BirA*_GGS_SNAP-tag and cMyc_PTP_SNAP-tag systems showed promising results, effectively isolating targets like DHFR-TS and TbAK, related to methotrexate and the C1 molecule.

In conclusion, this effort resulted in a promising target detection and capture technology for discovering potential targets of compounds in T. brucei and other parasites.

Title	Published in	Access level	OA Policy	Year	Views	Downloads

A novel tool for small molecule target identification in cytosolic and organellar sub-proteomes of Trypanosoma under in vivo conditions				2023	127	141

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Sahraoui, Suzanne Sherihan

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