期刊
CHEMISTRY-A EUROPEAN JOURNAL
卷 28, 期 62, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202201636
关键词
activity-based probes; affinity chromatography; cysteine proteases; imaging agents; peptidomimetics; rhodesain; size exclusion chromatography
资金
- BIGS DrugS graduate school of the University of Bonn
- German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes)
- project ChemBioDrug from ERDF/OPRDE [CZ.02.1.01/0.0/0.0/16_019/0000729]
- Czech Ministry of Education [OPVVV16_019/0000759]
- Projekt DEAL
- [RVO 61388963]
We developed a bimodal rhodesain probe that can detect and quantify rhodesain with high sensitivity, and used it for inhibitor screening. This probe provides a new tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.
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.
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