An Activity-Based Probe for Cathepsin K Imaging with Excellent Potency and Selectivity

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.