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

Von Wiley-VCH zur Verfügung gestellt

A highly potent, bimodal activity-based probe for the therapeutically relevant cysteine protease rhodesain was developed. We utilized two different access points at the coumarin fluorophore for incorporation of the rhodesain inhibitor part and the biotin affinity label. The two reporter tags were employed for on-blot chemiluminescence detection and affinity purification as well as post-labeling quantification of the target protein by size exclusion chromatography with fluorescence detection.


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−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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