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An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity
Carina Lemke1, Jakub Benýšek2,3, Dominik Brajtenbach1
1Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, Bonn 53121, Germany.
Abstract:
The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.
Insights
Researchers developed novel fluorescent probes to target and detect cathepsin K, a key enzyme in bone diseases. These probes enable sensitive visualization and imaging, aiding in understanding bone turnover and related pathologies.
Area of Science:
- Biochemistry
- Molecular Biology
- Medicinal Chemistry
Background:
- Cathepsin K is a cysteine protease crucial for bone matrix degradation and implicated in bone diseases.
- Targeting cathepsin K offers a therapeutic strategy for conditions characterized by high bone turnover.
Purpose of the Study:
- To design and synthesize novel fluorescent activity-based probes (ABPs) for cathepsin K.
- To characterize the binding and inhibitory properties of these probes.
- To evaluate their utility in detecting and visualizing cathepsin K in biological systems.
Main Methods:
- Design and synthesis of fluorescent ABPs and their precursors.
- Biochemical assays to determine probe potency (k_inac/K_i) and selectivity.
- X-ray crystallography to elucidate the binding mode of probes with cathepsin K.
- Cell-based assays including live cell imaging of human osteosarcoma cells.
Main Results:
- ABP 25 demonstrated high potency and selectivity for human cathepsin K.
- Crystal structures revealed the binding interactions of ABP 25 and precursor 21 with cathepsin K.
- Probe 25 enabled sensitive detection and selective visualization of cathepsin K in complex proteomes.
- Live cell imaging confirmed the probe's applicability in a pathophysiological context.
Conclusions:
- Novel acrylamide-based Michael acceptor probes were developed for cathepsin K.
- ABP 25 is a potent and selective inhibitor and imaging agent for cathepsin K.
- These probes show promise for diagnostic and research applications in bone-related diseases.
