Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain

0561246 - ÚOCHB 2023 RIV DE eng J - Journal Article
Lemke, C. - Jílková, Adéla - Ferber, D. - Braune, A. - On, A. - Johe, P. - Zíková, Alena - Schirmeister, T. - Mareš, Michael - Horn, Martin - Gütschow, M.
Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain.
Chemistry - A European Journal. Roč. 28, č. 62 (2022), č. článku e202201636. ISSN 0947-6539. E-ISSN 1521-3765
R&D Projects: GA MŠMT(CZ) EF16_019/0000729; GA MŠMT(CZ) EF16_019/0000759
Institutional support: RVO:61388963 ; RVO:60077344
Keywords : activity-based probes * affinity chromatography * cysteine proteases * imaging agents * peptidomimetics * rhodesain * size exclusion chromatography
OECD category: Biochemistry and molecular biology; Microbiology (BC-A)
Impact factor: 4.3, year: 2022
Method of publishing: Open access
https://doi.org/10.1002/chem.202201636

Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a k(inac)/K-i value of 37,000 M(-1)s(-1). Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.
Permanent Link: https://hdl.handle.net/11104/0333931