Two Tags in One Probe: Combining Fluorescence- and Biotin-based Detection of the Trypanosomal Cysteine Protease Rhodesain |
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Authors: | Carina Lemke Dr. Adéla Jílková Dominic Ferber Dr. Annett Braune Anja On Dr. Patrick Johe Dr. Alena Zíková Prof. Dr. Tanja Schirmeister Dr. Michael Mareš Dr. Martin Horn Prof. Dr. Michael Gütschow |
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Affiliation: | 1. Pharmaceutical Institute, Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany;2. Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, 16610 Prague, Czech Republic;3. Research Group Intestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany;4. Institute of Pharmaceutical and Biomedical Sciences (IPBS), Johannes Gutenberg University of Mainz, Staudingerweg 5, 55128 Mainz, Germany;5. Biology Centre CAS, Institute of Parasitology, University of South Bohemia, Faculty of Science, Branišovská 1160/31, 37005 České Budějovice, Czech Republic |
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Abstract: | 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 kinac/Ki value of 37,000 M−1s−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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Keywords: | activity-based probes affinity chromatography cysteine proteases imaging agents peptidomimetics rhodesain size exclusion chromatography |
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