Journal of the American Chemical Society
Article
behave autonomously, with respect to cargo degradation and
antigen presentation as a result of differential phagosome
maturation.41 This finding could explain the differential
cathepsin compartmentalization in specialized vesicular com-
partments. The confirmation and further understanding of this
phenomenon will be the basis for further investigation. Overall,
these observations highlight the potential of the near-infrared
cathepsin S selective qABP in combination with complemen-
tary pan-reactive cysteine cathepsin qABPs for activity local-
ization studies in living cells and animal models.
AUTHOR INFORMATION
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Corresponding Authors
Present Address
#Department of Chemistry, Yale University, New Haven, CT,
United States
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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CONCLUSIONS
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This work was supported by NIH grants R01 EB005011, R01
HL116307 (to M.B.), The Netherlands Organization for
Scientific Research (NWO) Rubicon fellowship (to M.V. and
W.A.v.d.L.), NIH grant R01 GM054051 (to J.A.E.), and NIH
Re-entry into Biomedical Sciences Supplement
3R01EB005011-06S1 (to K.O.B). C.G.F is recipient of an
NWO Spinoza award, ERC advanced grant PATHFINDER
(269019), and KWO award KUN2009-4402 from the Dutch
Cancer Society. We thank the members of the Bogyo lab and
the Figdor lab for insightful discussions, T. Doyle at the
Stanford Small Animal Facility, and S. Lynch at the Stanford
NMR Facility. We thank Ben Joosten for assistance with
CLSM, Josh Lichtman, and Josh Elias for recording HRMS data
and A. Chien and T. McLaughlin at the Stanford Mass
Spectrometry Facility for their technical assistance.
The cysteine cathepsin family members, once thought to only
be responsible for degradation within lysosomal compartments,
are slowly revealing their diverse functional roles in numerous
physiological processes. As their role in early development and
progression of various human pathologies is becoming more
evident, the cysteine cathepsins are increasingly being validated
as viable therapeutic targets. Due to its distinctive expression
profile and direct involvement in antigen presentation,
cathepsin S has attracted significant attention from scientists,
both in the pharmaceutical industry and in academia. In order
to investigate the mechanisms important for cathepsin S-
mediated pathology, reliable molecular tools that can monitor
cathepsin S activity are needed. These tools are important not
only to demonstrate the value of cathepsin S activity for
diagnosis and as a biomarker for disease progression but also to
develop and characterize novel therapeutic inhibitors or disease
modulators. Several cathepsin S inhibitors are currently in
various phases of clinical trials42 (Eli Lilly Inc., Virobay Inc.).
Building on our experience in the design and synthesis of
cysteine protease ABPs, we describe here a highly selective,
non-peptidic cathepsin S near-infrared qABP BMV157. This
probe can be used for sensitive, selective, and quantitative
assessment of cathepsin S activity in cells and even in whole
organisms. Complementary to this near-infrared cathepsin S
selective imaging tool, we synthesized and characterized a
green-fluorescent pan-cysteine cathepsin qABP EM053, which
we used in combination with BMV157 to visualize localization
of cathepsin S activity relative to the activity of cathepsin X, B,
and L in living cells. Importantly, the power of our
complementary chemical toolkit was demonstrated by the
observation that, unlike BMMs, BMDCs contain a pool of
vesicles that exclusively possess cathepsin S activity. This work
therefore provides valuable tools that can be used to determine
the characteristics of these distinct subcellular compartments.
While it remains unclear how and why such cathepsin S-specific
vesicles exist, they may play important roles in DC immunology
and provide insight into how this professional APC functions in
vivo. Overall, our results highlight the potential of the near-
infrared cathepsin S selective qABP and its combination with
complementary pan-reactive cysteine cathepsin qABPs for in
depth activity localization studies.
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ASSOCIATED CONTENT
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* Supporting Information
Experimental details of the synthesis of all probes and biological
experiments, supplemental figures, NMR spectra, and a video
file. This material is available free of charge via the Internet at
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX