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

Diplôme Thèse A novel tool for small molecule target identification in cytosolic and organellar sub-proteomes of Trypanosoma under in vivo conditions eng 2023 2023 author Sahraoui Suzanne Sherihan 795651 director Scapozza Leonardo 600318 Section des sciences pharmaceutiques (FARMA) 249 10.13097/archive-ouverte/unige:171172 urn:nbn:ch:unige-1711724 Sc. Vie - Pharma. 229 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. Human African Trypanosomiasis T. brucei SNAP-tag BioID BirA* Target detection and capture technology cMyc_BirA*_GGS_SNAP-tag cMyc_PTP_SNAP-tag DHFR-TS TbAK Dewey 615 31 Section des sciences pharmaceutiques 17 false 880 Biochimie pharmaceutique a2fab6db-82fc-4eda-b771-2a5d6055b5b1 Thesis Public Thèse Finale_Suzanne Sherihan Sahra.pdf 17930270 application/pdf 0f0c2fdb91dcb973244721cedbd1fbd968f6c9475607dbee32209f09d3e9ae93 7787951d-1fa7-4694-a249-0f9c5cc553a5 Imprimatur Public Imprimatur-1.pdf 67493 application/pdf 86c5dd4e1af740b3109af8148371565aa70497611ea8f90a79a11e0167986fb6 openAccess Faculté des sciences / Section des sciences pharmaceutiques

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A novel tool for small molecule target identification in cytosolic and organellar sub-proteomes of Trypanosoma under in vivo conditions				2023	360	438

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Sahraoui, Suzanne Sherihan

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