Cell penetrable, clickable and tagless activity-based probe of human cathepsin L

Article abstract of DOI:10.1016/j.bioorg.2019.02.032

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. I

Full text of DOI:10.1016/j.bioorg.2019.02.032

Accepted Manuscript
Cell Penetrable, Clickable and Tagless Activity-based Probe of Human Cathe-
psin L
Dibyendu Dana, Jeremy Garcia, Ashif I. Bhuiyan, Pratikkumar Rathod, Laura
Joo, Daniel A. Novoa, Suneeta Paroly, Karl R. Fath, Emmanuel J. Chang, Sanjai
K. Pathak
PII:
S0045-2068(18)31378-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.032
YBIOO 2807
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
28 November 2018
12 January 2019
12 February 2019
Please cite this article as: D. Dana, J. Garcia, A.I. Bhuiyan, P. Rathod, L. Joo, D.A. Novoa, S. Paroly, K.R. Fath,
E.J. Chang, S.K. Pathak, Cell Penetrable, Clickable and Tagless Activity-based Probe of Human Cathepsin L,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.032
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Cell Penetrable, Clickable and Tagless Activity-based Probe of
Human Cathepsin L
Dibyendu Dana,1,2 Jeremy Garcia,3 Ashif I. Bhuiyan1,6, Pratikkumar Rathod,4,7 Laura Joo,1 Daniel A.
Novoa,1 Suneeta Paroly,5 Karl R. Fath,3,6 Emmanuel J. Chang,4,2,6 Sanjai K. Pathak1,2,6,*
1Queens College of The City University of New York, Chemistry and Biochemistry Department, 65-30 Kissena Blvd, Flush-
ing, NY 11367, United States; 2Chemistry Doctoral Program, The Graduate Center of The City University of New York, 365
5th Ave, New York, NY, 10016, USA.; 3Queens College of The City University of New York, Department of Biology, 65-30
4
Kissena Blvd, Flushing, NY 11367, USA.; York College of The City University of New York, Department of Chemistry,
5
94-20 Guy R. Brewer Blvd, Jamaica, NY, 11451-0001, USA.; Bard High School Early College Queens, 30-20 Thomson
6
Avenue, Long Island City, USA; Biochemistry Doctoral Program, The Graduate Center of The City University of New
York, 365 5th Ave, New York, NY, 10016, USA.; 7Laguardia Community College, 31-10 Thomson Ave, Long Island City,
NY 11101, USA
*Correspondence: Sanjai.Kumar@qc.cuny.edu; Phone: +1 718 997 4120; Fax: +1 718 997 5531
KEYWORDS Activity-based probe, cathepsin L probe, copper-mediated azide-alkyne cycloaddition, clickable probe, tagless
cathepsin L probe
ABSTRACT: Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of
cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of
cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even
opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in
context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clicka-
ble and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-
dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can in-
hibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dis-
secting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-
discovery
efforts
targeting
cathepsin
L.
tional roles were often considered redundant. Recent studies,
however, strongly point to individual members of cysteine
cathepsins having a specific and non-redundant function in
various cell types.4,5 They can regulate specific physiological
processes such as antigen presentation in the immune response
pathway, neuropeptide and hormone processing, apoptosis,
autophagy, and bone development and remodeling.6-9 Consist-
ently, their localization is not limited to only in the endo-
lysosomal compartment; they are also found in other intracel-
lular organelles such as cytosol, nucleus, secretary vesicles,
and in the extracellular matrix. The substrate preferences of
various cathepsins using a large synthetic library as well as
cellular proteome have revealed that both a relatively broad
tolerance and a stricter preference exist for amino acids at
distinct positions around the scissile peptide bond.10,11 One of
the main challenges in cathepsin biology remains to assign
1. Introduction:
Cellular proteolysis is a highly dynamic, complex, and yet
well-organized event orchestrated by more than 550 human
proteases.1,2 The basic process involves catalytic hydrolysis of
a specific peptide bond of protein targets under a wide range
of cellular conditions (e.g. pH, specific organelle, cytoplasmic
vs extracellular). These enzymes play critical roles in main-
taining homeostasis, growth, differentiation, and survival.
Among these are cysteine cathepsins, a small group of 11
proteolytic enzymes that usually function in acidic cellular
environment (e.g. in endosomes/lysosomes), and employ a
Cys-dependent covalent nucleophilic catalysis. Originally,
cysteine cathepsins were considered a group of house-keeping
enzymes whose primary function was to merely degrade un-
wanted cellular proteins in lysosomes.3 As a result, their func-

function to individual members of cysteine cathepsins. This
task is especially difficult since cathepsins are initially synthe-
sized as zymogens (inactive enzyme), and stored in special-
ized organelles. They are often activated by post-translational
modifications (e.g. proteolysis, glycosylation and phosphory-
lation), and transported to various cellular compartments for
function.12 For such reasons, robust functional assignment of
enzymes such as cathepsins have proven challenging, and
have led to datasets where estimate of cellular enzyme activi-
ties have been off by orders of magnitude13-15. Other complexi-
ties also exist in defining specific cathepsin function because
of the existence of (a) endogenous cysteine cathepsin inhibi-
tors (e.g. cystatins and stefins), and (b) existence of alternative
splice variants of enzymes, and overlapping substrate specific-
ities.
is substrate-based probes (e.g. compound 1 and 2, see Figure
1). They are turned over like a substrate at a specific amide
bond (in blue) producing enhanced fluorescence readout. The
fluorescence enhancement is either due to a relief of quench-
ing mechanism (probe 1), or by production of a highly fluo-
rescent chemical moiety (probe 2) upon turnover.64,65 Alt-
hough substrate-based probes produce an activity-dependent
fluorescence readout, they often suffer from few major limita-
tions: They do not covalently label their target cathepsins; thus
it becomes almost impossible to conclude if the optical signal
is directly coming from the active intracellular target
cathepsin. This becomes especially an issue when overlapping
substrate specificities exists, making the quantification of in-
tracellular activity problematic for precise functional assign-
ments. Finally, they can be bulky in nature (hence reduced
affinity), and often possess high charge density (hence poor
cell penetrability). The second category of probes are
photoaffinity probes which have chemical functionalities suit-
able for a reversible binding to their target enzymes, and con-
tain an additional photoactivatable group. These probes can be
activated for a covalent bond formation with their target en-
zymes upon exposure to photons. An example of this kind of
probe is probe 3 (Figure 1), developed for monitoring
cathepsin L activity. Unfortunately this probe also contains a
bulky photoactivatable benzoylphenylalanyl group that ren-
ders it only modestly potent (Ki = 3.7µM), and poorly selective
(against cathepsins B).66 Finally, such probes may also suffer
from non-specific labeling of surrounding proteins, often due
to less-than-desirable affinity.83,84 The third category of probes
are ABPs which exploit a conserved catalytic residue of the
target cathepsin (the key nucleophilic catalytic residue Cys25),
thereby labeling the enzyme covalently and irreversibly in an
activity-dependent manner. Probe 4A and 4B (Figure 1) are
two examples of such cysteine cathepsin-targeted activity-
based probe (ABP). The probe 4A’s response to the cellular
activity of cathepsin L enzyme relies on relief of the quench-
ing mechanism (as in probe 1) (thus the name qABPs) of the
installed fluorophore.68,69,79 While several highly interesting
findings have recently emerged from studies involving qABPs,
these probes suffer from less-than-desirable selectivity toward
cathepsin L (with respect to cathepsin B) and sensitivity. Us-
ing an elegant positional scanning substrate approach,
HyCoSuL, Poreba et al. developed an efficient activity-based
cathepsin L-selective probe 4B; however this probe also con-
tained a bulky (molecular weight = 1410) and charged Cy5
fluorophore, and exhibited only 412-fold selectivity over
cathepsin B.85 Herein, we report the development of a new
class of clickable and tagless activity-based probe (ctABP) of
human cathepsin L. We show that the probe is ~10,000-fold
selective toward cathepsin L (over cathepsin B), and demon-
strate its utilization in probing cathepsin L activity in human
MDA-MB-231 breast cancer cell lines. Furthermore, we
demonstrate that that this probe can be utilized for perturba-
tion of previously reported cathepsin L function in vivo.
A prominent member of the cysteine cathepsin pro-
tease family is cathepsin L, which is involved in a variety of
critically important cellular processes.5,9,16-28 Aberrant expres-
sion and activation of cathepsin L has been implicated in many
types of human diseases.5,18,29-46 These include promoting
highly invasive cancer, cardiovascular, lung, immune, and
metabolic disorders.4,28,41,42,47-51 In addition to its involvement
in human biology, cathepsin L has been shown to promote
deadly infections arising from viral and parasitic pathogens;
some of these include, Ebola, and Severe Acute Respiratory
34
Syndrome (SARS) viruses.52
Despite cathepsin L’s in-
volvement in several human diseases, many of its function in
appropriate cellular contexts (i.e. different cell types) remain
poorly understood.5,16,18,53 This problem has been further exac-
erbated since both contradictory and redundant roles of vari-
ous cathepsin enzymes have recently emerged. For example,
while cancer-promoting roles of cathepsin L are well docu-
mented, recently in mouse model cathepsin L served as an
epidermal tumor suppressor by terminating the growth factor
signaling.54 In another example, cathepsin L seems to be in-
volved in both bone growth and bone loss processes.55,56 An-
other challenge related to functional dissection of cathepsin L
is due to presence of an analogous cathepsin B which also
shares redundant as well as contradictory function. For exam-
ple, in the inflammatory response signaling pathways, both
cathepsin B and L can serve in a mutually compensatory role
during the synthesis and activation of the key cytokine mole-
cule, IL-1β.57 In contrast, processes involved in regulating the
release of digestive proteases from pancreas for food diges-
tion, cathepsin B and L seem to play opposing (and non-
redundant) roles; while cathepsin B activates the key protease,
trypsin, for initiating the digestive proteolysis cascade58,59
,
cathepsin L seem to function so as to keep trypsin activation in
check.60 Similarly, in primary hippocampal neuronal signaling
during the amyloid precursor protein processing by γ-
secretase, cathepsin B elevated while cathepsin L decreased
the γ-secretase activity.61 Cathepsin L is postulated to be a
homeostatic protease in cardiac signal transduction but what,
if any, role does cathepsin B play in these events remains an
open question.62,63 Small molecule cathepsin L probes that can
selectively (over cathepsin B) report on cathepsin L activity in
cells with high sensitivity can thus help expedite functional
assignments of cathepsin L in both normal biology and dis-
eased states.
2. Results and Discussion:
2.1 Rationale for the Design: The motivation for
the current work stemmed from our hypothesis that develop-
ment of a cathepsin L ABP with a strategically-positioned
small clickable moiety without bulky and charged functional
groups (e.g. fluorophore tag and quencher moiety) could pro-
vide a highly effective probe with enhanced cell penetrability,
Several small molecule probes of cysteine cathepsins
have been developed in the recent years.64-82 These probes can
broadly be classified into three major categories. The first type

higher affinity and selectivity and sensitivity of detection.
Such clickable and tagless activity-based probe (ctABP) could
prove to be highly useful for monitoring activity profile of
human cathepsin L in living cells at the proteome levels. Re-
cently using a hybrid-design approach, we reported the devel-
opment of a highly potent, cell permeable, and non-basic in-
hibitor, KD-1, of human cathepsin L (Figure 2).86 KD-1’s non-
basic nature was especially important so that homogenous
distribution of inhibitor in the cell can be accomplished; else
an undesired accumulation would occur in acidic environment
of the lysosome.87-89 This inhibitor was active-site directed,
rapidly inhibited cathepsin L activity in a covalent and irre-
versible manner, and exhibited very high selectivity preference
over cathepsin B.86 This could therefore serve as an excellent
starting point for acquiring cathepsin L ABP. Since the
carbobenzyloxy (cbz) group of KD-1 likely sat in the S3 pock-
et and knowing that S3 pocket of cathepsin L could accommo-
date bulkier aromatic groups, we thought it possible to incor-
porate a minimally perturbing small clickable ethynyl group
on the cbz moiety.10 The main advantages of this design ap-
proach were envisioned to be 2-fold: (i) The neutral ethynyl
group inclusion, while anticipated to minimally affect the ki-
netic parameter of cathepsin L inactivation, was designed to
retain or perhaps augment the cell penetrability, and (ii) The
ethynyl group would prove to be a highly valuable tool in de-
tection of labelled cathepsin L from living cells using aque-
ous-friendly well-established click chemistry protocols.90 This
led to the conception of a ctABP of cathepsin L, KDP-1 (Fig-
ure 2).
Cys containing protein tyrosine phosphatase 1B enzyme, and
the serine protease trypsin.
2.3 Mass Spectrometry-based Characterization of
Inhibition: To establish that active site Cys25 was the target
for covalent labeling, mass spectrometric analysis was per-
formed on human cathepsin L labelled with KDP-1 probe
(Figure S2, Supporting Information). The MS2 spectrum at m/z
= 2230.7 from a chymotryptic digest of KDP-1-reacted
Cathepsin
L,
corresponding
to
residues
16-30
(16VTPVKNQGQCGSCWA30), contains major peaks con-
sistent with neutral loss of KDP-1-related fragments, as well
as an N-terminal backbone fragment with the label still at-
tached (b*12, cleavage between Ser27 and Cys28). This latter
fragment indicates that the modification is at Cys25 rather
than Cys28. These experiments together indicated that the
developed cathepsin L ctABP is a selective active-site directed
probe.
2.4 Activity-dependent Labeling of Human
Cathepsin L: The next task in hand was to demonstrate if (a)
KDP-1 was a suitable probe for labeling active human
cathepsin L, (b) the ethynyl moiety was amenable for post-
labeling quantification of active enzyme using click chemistry
protocol, and (c) the enzyme labeling reaction was active site-
directed, covalent and irreversible in nature. To investigate
this, active recombinant human cathepsin L was incubated
with KDP-1 probe in an acidic buffered solution. After the
labeling, a click chemistry protocol was performed using
TAMRA-azide. A varying amount of TAMRA-KDP-1-
cathepsin L complex was resolved using SDS-PAGE and the
gel was analyzed using Typhoon 9400 scanner. The scanning
experiment revealed that as low as 40 femtomoles (1 ng) of
labeled active cathepsin L could be detected with this probe
(Figure 4A), making it a highly sensitive probe, suitable for
cellular studies; a covalent and irreversible nature of labeling
was also established from this experiment. No labeling was
observed when heat-denatured cathepsin L was allowed to
react with KDP-1 and undergo identical click chemistry pro-
cedure (Figure 4B left); this experiment indicated an activity-
dependent labeling. Finally, when active cathepsin L reaction
with KDP-1 was performed in presence of a competitive in-
hibitor, a significantly reduced amount of labeling was ob-
served, indicating active-site directed chemistry of labeling.
2.2 Chemical Synthesis and Steady State Enzy-
mology of Inhibition Kinetics: The probe KDP-1 was syn-
thesized as outlined in Scheme 1.91 Briefly, this synthesis
started with construction of (((4-ethynylbenzyl)oxy)carbonyl)-
L-phenylalanine intermediate 8 in three steps, starting from
commercially available (4-ethynylphenyl)methanol. Interme-
diate 8 was then condensed with ethyl(S,E)-3-amino-5-
methylhex-1-ene-1-sulfonate to generate the vinylsulfonate
ethyl ester intermediate 9. Deprotection of ethyl group fol-
lowed by reaction with 4-bromophenol yielded KDP-1. The
next step was to assess if the time-dependent inhibition kinet-
ics of KDP-1 probe with human cathepsin L was still robust,
and if the developed probe maintained its selectivity profile.
Steady state enzymology experiments revealed that KDP-1
exhibited a strong time dependent inactivation of cathepsin L
activity, although with ~10-fold lower rate (Figure 3) than
KD-1; this perhaps could be due to some unfavorable interac-
tion within the S3 pocket of cathepsin L enzyme and may re-
quire structural studies in future. Furthermore, KDP-1 still
maintained a strong selectivity preference for cathepsin L
compared to cathepsin B (~10,000 fold) and homologous
cathepsin K (~29-fold), and displayed no reactivity towards
cathepsin H, also a cysteine cathepsin. A ~10,000-fold
Cathepsin L selectivity over cathepsin B is especially notewor-
thy since the probe can potentially be utilized for sorting func-
tional redundancies between the two in diverse cellular signal-
ing events; a recently reported human cathepsin L ABP only
exhibited ~412-fold selectivity over human cathepsin B.85
Aspartyl cathepsin D and serine cathepsin G also exhibited no
noticeable reactivity toward KDP-1, indicating the absolute
requirement of a suitably positioned active site Cys25 residue.
Further, no cross reactivity was observed against a low pKa
2.5 Assessment of Cytotoxicity, Cell-penetrability,
and Cell-migratory Potential Using Triple-Negative Hu-
man MDA-MB-231 Cell Lines: Efficient activity-based
probes can be a very valuable tool for functional studies as
well as for assessing in vivo target engagement of a
drug/probe92,93 It is therefore imperative that they be non-toxic
and biocompatible. Since future studies will involve the use
of KDP-1 in cellular studies, we investigated its toxicity in cell
culture using human MDA-MB-231 breast cancer cells and the
CytoScan WST–1 cell viability assay (G-Biosciences Inc.).94
This assay is based on determining metabolically active cells
by monitoring reduction of tetrazolium salt WST-1 to
formazan by cellular dehydrogenases. No apparent cellular
toxicity was observed, even as high as 10 µM of the probe
(Figure 5A). Ubiquitously expressed human cathepsin L has
been shown to have several cytoplasmic and nuclear function,
in addition to its roles in extracellular matrix (ECM)
space.5,95,96 To demonstrate that developed cathepsin L
ctABP, KDP-1 was capable of labeling intracellular cathepsin

L in living cells, we sought to establish that it is cell permea-
ble. Human MDA-MB-231 breast cell line was chosen for this
study since herein cathepsin L is known to be overex-
pressed.97,98 Live cells were treated with KDP-1 probe (with
DMSO as negative control) and cells were washed and
permeabilized under non-denaturing conditions. Cellular
cathepsin activity was measured using the Z-Phe-Arg-AMC
fluorogenic cysteine cathepsin substrate. A dose dependent
loss of activity was observed, thereby indicating that KDP-1
was cell penetrable and was likely able to block cellular
cathepsin L activity (Figure 5B). In positive control experi-
ments, cells treated with cell permeable cathepsin L inhibitor
KD-1 and pan cysteine cathepsin inhibitor E-64d also exhibit-
ed significantly diminished cathepsin L activity.86,99,100 These
studied together suggest that KDP-1 is a nontoxic probe, and
can be utilized to label active cellular cathepsin L at the prote-
ome level in the living cells from diverse origin.
The development of a highly efficient, cell-penetrable and
clickable probe (KDP-1) of human cathepsin L is reported.
Steady-state enzymology, labeling, and mass-spectrometric
experiments indicate that the probe is highly sensitive, selec-
tive, and active-site directed. Cell-biology experiments involv-
ing human MDA-MB-231 breast cancer cell lines reveal that
the probe is non-toxic, can block cathepsin L activity, and
reduce cell migratory phenotype. Finally using live zebrafish
embryos, we demonstrate that presence of KDP-1 can delay
hatching – a biological process where cathepsin L activity is
required. We anticipate that this ctABP cathepsin L probe will
find extensive applications in both functional proteomics and
in cathepsin L-targeted drug discovery processes.
4. Experimental:
4.1 Organic Synthesis:
4.1.1 General: 1H NMR spectra were recorded at ei-
We further investigated if the developed probe,
KDP-1 was biologically relevant; i.e. if it could successfully
perturb cathepsin L function in cellular settings and in vivo. In
several biological processes involving ECM remodeling,
cathepsin L plays important roles.23,101-103 For example in high-
ly metastatic cancers, overexpressed cathepsin L promotes cell
motility by degrading the adherens junction protein, E-
cadherin.4 To investigate if indeed the inhibitory KDP-1 probe
reduced the migratory phenotype promoted by cathepsin L, we
carried out experiments to assess cell motility in live cells. A
wound-healing assay was thus performed on highly motile
human MDA-MB-231 breast cancer cells (Figure 6). Com-
pared to cell incubated in solvent control (0.5% DMSO), cells
incubated with 1.25µM KDP-1 had a 5% decrease in cell mi-
gration rate, while 3.75µM KDP-1 reduced the migration rate
20% (independent samples two-tailed t-test pobs= 0.15). This
dose-dependent reduction in cell motility is consistent with the
inhibition of cathepsin L in ECM space, and supports the no-
tion that KDP-1 can indeed be utilized to probe biological
function of cathepsin L in distinct cellular environment.
ther 400 or 500 MHz using CDCl3 or DMSO as the solvent.
13C NMR spectra were recorded at either 125 or 100 MHz
using CDCl3 or DMSO as the solvent. Chemical shifts (δ) are
reported in parts per million (ppm) and referenced to CDCl3
(7.26 ppm for 1H and 77.0 ppm for 13C), DMSO (2.50 ppm for
1H and 39.5 ppm for 13C). Coupling constants (J) are reported
in Hertz (Hz) and multiplicities are abbreviated as singlet (s),
doublet (d), doublet of doublets (dd), triplet (t), triplet of dou-
blets (td), and multiplet (m). The mass spectra were acquired
using a 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-
TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA).
4-ethynylbenzyl alcohol was purchased from Santa Cruz Bio-
technology, Inc. (Dallas, Texas, USA).
O-(1H-6-
Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HCTU) was purchased from peptide
international (Louisville, Kentucky, USA). All anhydrous
solvents were purchased from Sigma-Aldrich (St. Louis, MO,
USA). All other materials were purchased from Fisher Scien-
tific Inc. (Pittsburgh, PA, USA) unless otherwise mentioned in
the text.
Synthesis Protocol: The Synthesis of the clickable
and tagless activity-based probe (ctABP), KDP-1 was
achieved following the scheme outlined in Scheme 1:
2.6 Demonstration of Functional Perturbation of
Cathepsin L Activity In Vivo by Cathepsin L Probe, KDP-
1: in Finally, the inhibitory effect of KDP-1 in vivo was in-
vestigated in a zebrafish system. The hatching process in
zebrafish involves a major tissue remodeling. To carry out this
function, overexpressed cathepsin L in hatching gland is se-
creted to degrade proteinaceous chorion.104,105 Activity of
cathepsin L is thus required for the hatching process to begin,
and serves as an excellent marker for studying mesoderm and
axis induction during the hatching process. A group of
zebrafish embryos post-fertilization were treated with KDP-1
at appropriate concentrations and embryos hatched at different
hours post-fertilization (hpf) were counted. A dose-dependent
decrease in hatching was observed between 49-51 hpf com-
pared to control (DMSO) (Figure 7 II). In positive control, a
high concentration of pan cysteine cathepsin inhibitor E64 (10
µM) delayed the hatching process considerably with only 50%
hatching, even at 54-72 hpf. This experiment further demon-
strates that the developed probe KDP-1 is amenable for study-
ing cathepsin L-mediated in vivo function.
(i) 4-ethynylbenzyl carbonochloridate (6): The reaction was
carried out by following a literature supported protocol.106
First, 2.25 g (7.57 mmol, 2 equiv) of triphosgene was dis-
solved in a minimal amount of anhydrous toluene in the pres-
ence of 1.88 g (17.7 mmol, 9 equiv) of oven dried sodium
carbonate (Na2CO3) under inert atmosphere at 0°C. After al-
lowing the mixture to stir for 35 min, 0.500 g (3.78 mmol, 1
equiv) of 4-ethynylbenzyl alcohol dissolved in dry toluene was
added dropwise. Then, the mixture was stirred overnight at
room temperature. The reaction mixture was filtered, evapo-
rated, and the resulting 4-ethynylbenzyl carbonochloridate was
used without purification.
(ii) Methyl (((4-ethynylbenzyl)oxy)carbonyl)phenylalaninate
(7): The reaction was carried out by following a literature re-
ported protocol with slight modification.107 To a stirred sus-
pension of L-phenylalanine methyl ester hydrochloride (815
mg, 3.8 mmol, 1 eqv.) in 5 ml of toluene was added 4-
ethynylbenzyl carbonochloridate (735.6 mg, 3.8 mmol, 1 eqv.)
in an icebath. To this was added 5.3 ml of 1 M sodium car-
bonate solution. The reaction mixture was kept stirring for 3
3. Conclusion:

hrs while the temperature was maintained at less than 10°C.
The reaction mixture was diluted with ethyl acetate and
washed with 0.1 M HCl, saturated sodium bicarbonate solu-
tion and brine successively. The organic layer was dried over
sodium sulfate and purified by silica gel column chromatog-
raphy using hexanes and EtOAc in a 95:5 ratio, yielding a
white solid (Rf = 0.44, Hexanes/EtOAc (80:20), 46.3% yield).
The characterization was found to be consistent with the pre-
viously reported NMR spectra.108
Lys(Dnp)-D-Arg-NH2 (BML-P145), Z-Val-Val-Arg-AMC
(BML-P199).
(BAPNA)
N-alpha-Benzoyl-DL-arginine-p-nitroanilide
(B4875), trypsin (T1426), para-
nitrophenylphosphate were purchased from Sigma–Aldrich
(St. Louis, MO, USA). All the biological grade buffers were
used and were purchased from Sigma–Aldrich (St. Louis, MO,
USA). All enzyme kinetics experiments (unless otherwise
stated) were carried out at 30°C in appropriate buffer condi-
tion with 5% DMSO concentration. For measuring the initial
rates of enzyme catalyzed reaction, a temperature-controlled
steady-state arc lamp fluorometer equipped with Felix 32
software (Photon Technology Instrument, Birmingham, NJ,
USA) and a UV–Vis spectrophotometer (Modelk25; Perkin
Elmer Inc., Waltham, MA, USA) was used.
(iii) (((4-ethynylbenzyl)oxy)carbonyl)-L-phenylalanine (8):
Subsequent deprotection to the carboxylic was carried out by
following the literature reported protocol and its characteriza-
tion was found to be consistent with the previously reported
NMR spectra.108
4.2.2 Determination of Second Order Inactivation
Rate Constant of KDP-1 Reactivity with Human Cathepsin
L and its Selectivity Profile: A previously reported cathepsin
L assay procedure was adopted with suitable modifications.86
Briefly, recombinant human liver cathepsin L (Enzo Life Sciences,
USA) was first activated by incubation in sodium acetate buffer
(50mM, pH 5.5) for 30 minutes at 30 °C. The inactivation reaction
at pH 5.5 was then continuously monitored for 3 minutes under
pseudo first order condition ([I]>>[E]; Net [E] 160 pM; [I] 5
nM – 100 nM) in a fluorescence (excitation:365 nm; emission:
440 nm) quartz cuvette containing Z-FR-AMC substrate (net 5
μM) maintained at 30°C. The progress curves thus obtained at
various KDP-1 concentration were fitted using a non-linear
regression method to the following equation:110
(iv) Ethyl (E)-3-amino-5-methylhex-1-ene-1-sulfonate: This
compound was synthesized by following a literature reported
protocol.109
(v)
Ethyl
(S,E)-3-((S)-2-((((4-
ethynylbenzyl)oxy)carbonyl)amino)-3-phenylpropanamido)-5-
methylhex-1-ene-1-sulfonate (9): This compound was synthe-
sized by following an analogous
literature reported
protocol.109 1H NMR (400 MHz CDCl3) δ ppm: 0.87 (m, 6H),
1.31 (m, 2H), 1.38 (t, J=7.0 Hz, 3H), 1.50 (m, 1H), 3.02 (dd,
J= 13.5 and 8.1 Hz, 1H), 3.12 (dd, J= 13.5 and 8.1 Hz, 1H),
3.09 (s, 1H), 4.14 (m, 2H), 4.36 (m, 1H), 4.62 (m, 1H), 5.08
(m, 2H), 5.33 (b, 1H), 5.75 (b, 1H), 5.94 (d, J=15.2 Hz, 1H),
6.60 (dd, J= 15.2 and 5.1 Hz, 1H), 7.18 (d, J=7.0 Hz, 2H),
7.30 (m, 5H), 7.48 (d, J=8.2 Hz, 2H). 13C NMR (100 MHz
CDCl3) δ ppm: 14.9, 21.8, 22.6, 24.6, 38.5, 42.7, 48.1, 56.6,
66.7, 66.9, 77.8, 83.2, 122.2, 124.6, 127.6, 127.9, 129.1,
129.2, 132.4, 135.9, 136.6, 147.6, 155.9, 170.4.
P = vz [1- exp(-kobs* t)] / kobs
where [P] is the product, vz is the initial velocity (at time zero),
and kobs is the pseudo-first order rate constant.
(vi)
4-bromophenyl
(S,E)-3-((S)-2-((((4-
The pseudo-first order rate constants kobs thus obtained from
the fit above were plotted against corresponding inhibitor con-
centrations [I]. The 2nd order enzyme inactivation rate con-
stant, kinact, was calculated (Figure S1, Supporting Infor-
mation) from the slope of the plot according to following
ethynylbenzyl)oxy)carbonyl)amino)-3-phenylpropanamido)-5-
methylhex-1-ene-1-sulfonate (KDP-1): This compound was
synthesized by following an analogous literature reported
protocol.109 1H NMR (400 MHz CDCl3) δ ppm: 0.82 (m, 6H),
1.22 (m, 2H), 1.41 (m, 1H), 2.99 (dd, J= 13.5 and 8.4 Hz, 1H),
3.10 (dd, J= 13.5 and 8.4 Hz, 1H), 3.10 (s, 1H), 4.37 (m, 1H),
4.53 (m, 1H), 5.06 (m, 2H), 5.44 (d, J=7.1 Hz, 1H), 5.89 (d,
J=5.2 Hz, 1H), 6.04 (d, J=15.1 Hz, 1H), 6.47 (dd, J= 15.1 and
5.3 Hz, 1H), 7.08 (m, 2H), 7.13 (m, 2H), 7.28 (m, 5H), 7.49
(m, 4H). 13C NMR (100 MHz CDCl3) δ ppm: 21.8, 22.5, 24.6,
38.4, 42.3, 48.2, 56.6, 66.7, 77.8, 83.2, 120.8, 122.2, 123.8,
124.4, 127.6, 127.9, 129.1, 129.1, 132.3, 132.9, 135.8, 136.5,
148.3, 149.9, 155.9, 170.5. HRMS (m/z): [M+H]+ for molecu-
lar formula C32H33BrN2O6S: calculated 653.1315; found
653.1318.
equation110
:
kobs = kinact [I] / [1+ ([S]/KM)]
where kobs is the pseudo-first order rate constant, [I] is the cor-
responding inhibitor concentration, [S] is the substrate concen-
tration with a given KM. KaleidaGraph (version 3.52) was used
for plotting and analyzing the data. All assays involving se-
lectivity profile of various enzymes were performed fol-
lowing the earlier published protocols.86,111,112
4.3 Tandem Mass-spectrometric Analysis of Hu-
man Cathepsin L Labelled with KDP-1:
4.2 Steady State Inactivation Kinetics:
4.2.1 General: The following enzymes and their
substrates were purchased from Enzo Life Sciences (Farming-
dale, NY, USA): cathepsin L (BML-SE201), cathepsin K
(BML-SE553), cathepsin B (BML-SE198), cathepsin D
A 10 µg KDP1-reacted and 5 µg unreacted human
Cathepsin L samples were run on a 4-12% % SDS-PAGE gel
and stained with colloidal coommassie blue (Thermo Scien-
tific). Bands were excised manually, and digested by sequenc-
ing grade chymotrypsin (Promega) at 27 °C for 6h. Chymo-
trypsin was added at the beginning of the digestion to a final
substrate-to-enzyme ratio of ~30:1 in a buffer containing 100
mM Tris, 10 mM CaCl2, pH 8.0 and supplemented at a sub-
strate-to-enzyme ration of ~60:1 halfway through the reaction.
After digestion, samples were prepared as previously de-
(BML-SE199), cathepsin
S (BML-SE453), cathepsin H
(BML-SE200), cathepsin G (BML-SE283), human PTP1B
(BML-SE332), Z-Arg-Arg-pNA (BML-P138), Z-Gly-Pro-
Arg-AMC (BML-P142), Z-Phe-Arg-AMC (BML-P139), H-
Arg-AMC.2HCl (BML-P135), Suc-Ala-Ala-Pro-Phe-pNA
(BML-P141),
Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-

scribed.113 Briefly, POROS 20 R2 resin (Applied Biosystems)
was added into the digested Cathepsin L samples for extrac-
tion at 4°C for 4h. Prior to MALDI mass spectrometric analy-
sis, the digests were desalted using ZipTip (Millipore) and
eluted from ZipTip resins directly onto a MALDI plate with a
saturated α-cyano-4-hydroxycinnamic acid (Sigma) MALDI
matrix solution trifluoroacetic acid/ acetonitrile /water
(0.1:70:29.9, v/v/v), and the solvent evaporated at room tem-
perature. The resulting MALDI crystals were analyzed by
hypothesis-driven MS2 at m/z values corresponding to poten-
tially-modified chymotryptic peptides (Figure S2, Supporting
Information).114,115 Mass spectrometry was performed using a
Thermo LTQ XL linear ion trap mass spectrometer (Thermo
Scientific) equipped with a vacuum MALDI source. MS2 spec-
tra were acquired using the supplied N2 laser (Thermo Scien-
tific) at a power of 5 µJ with a repetition rate of 10 Hz, using
an isolation width = 4.0 m/z, activation Q = 0.25 and activa-
tion time = 100 ms. Each spectrum was acquired for approxi-
mately 2.25 minutes. The resulting spectra were analyzed
manually with the assistance of PAWS sequence analysis
software (Genomic Solutions) and ProteinInfo (Rockefeller
University, NY; http://prowl.rockefeller.edu) to calculate
theoretical molecular weights.
acids, and glutamine). The next day, the medium was aspirated
from the cells and 100µl complete medium was added also
containing various concentrations of KDP–1 or 0.5% DMSO
(solvent control). After 24 hours of treatment, the relative
number of metabolically–active cells in each condition was
measured using the colorimetric CytoScan WST–1 cell prolif-
eration assay94 (G-Biosciences Inc., USA) in a microplate
reader. Average of three independent assays were taken into
consideration on separate days (biological replicates) and each
individual day assay was the average of 3 wells (technical
replicates). The degree of biocompatibility was calculated as
the percentage of cells alive in presence of KDP–1 relative to
the number alive in the presence of the solvent:
% survival = (ABS
– ABS
) / (
ABS cells +
KDP-1 no cells
cells + KDP–1
– ABS
)
DMSO
DMSO no cells
4.5.2 Assessment of Human Cathepsin L Activity
in MDA-MB-231 Live Cells Treated with KDP-1: The
method of Repnick was used.116 MDA MB–231 cells were
plated at 10–15 x 104 /well in complete medium cells in black
transparent bottom 96–well plates. The next day, fresh media
was added containing one of the following: 0.5% DMSO, 0.75
μM KDP-1, 10 μM KDP-1, 10 μM KD-1 or 10 μM E-64d (a
known broad range, cell-permeable cysteine protease inhibi-
tor). After 24 hours, soluble extracellular inhibitors were re-
moved by aspirating the media and washing the cells with
phosphate–buffered saline. Next a cellular lysate was made by
solubilizing the plasma membrane and intracellular mem-
branes by adding 50µl of 200 μg/mL digitonin in acetate buff-
er (50mM sodium acetate pH 5.6, 150 mM NaCl, 0.5 mM
EDTA) to the wells containing the cells and incubating on ice
for 12 minutes. We next added 50µl of the Omnicathepsin
fluorogenic substrate Z-FR-AMC in sodium acetate buffer
directly to the lysates in the wells for a final concentration of
30µM Z–FR–AMC and 10mM DTT. We quantified the rela-
tive amount of substrate cleavage in each well using
SpectraMax M4 multi-mode microplate reader at 380 nm exci-
tation and 460 nm emission. The experiments were repeated in
3 independent trials and each trial represented a technical rep-
licate of two. Because of slight variability in amount of sub-
strate or the number of cells in different trials, we normalized
the fluorescence generated in each trial relative to the DMSO
control from that trial. A one-way ANOVA was conducted to
compare the effect of the various inhibitors on intracellular
cathepsin. At 0.75 and 10µM KDP–1 and 10 µM of the inhibi-
tors KD-1, and E-64d, there was a significant inhibition of Z-
FR-AMC cleavage at the p < 0.05 level. Because these data
were proportional (percentages), significance was assessed by
first transforming the percentage data using an arcsine trans-
formation followed by a t Test with Bonferroni correction.
4.4 Labeling and Detection of Recombinant Ac-
tive Human Cathepsin L Using Click Chemistry: First, the
recombinant human Cathepsin L (2.2 µg, 5 µL of 17 µM sup-
plied stock, R&D Systems Inc.) was fully activated by incu-
bating for 30 min at 30 °C in 13.5 µL NaOAc buffer (50 mM,
pH = 5.5) containing 4mM DTT. Subsequently, cathepsin L
labeling was initiated with addition of 1.5 µL KDP-1 (net 25
µM in the total 20 µL reaction volume) and the labeling reac-
tion was allowed to occur for 2 h at 30 °C. To detect the
amount of KDP-1-labeled human cathepsin L, a click chemis-
try protocol was planned using 5-TAMARA azide (Click
Chemistry Tools, Inc., cat # 1245). The labeling reaction mix-
ture was brought to near neutral pH by addition of 2 µL
HEPES buffer (1000 mM, pH 8.0) before performing the click
chemistry reaction. To begin click chemistry the following
was added to the reaction mixture: 2 µL of TAMRA-Azide
solution (5mM stock in DMSO), 2 µL Sodium Ascorbate
(20mM stock in dH2O), 1 µL CuSO4 solution (10 mM stock in
dH2O), 1 µL THPTA (10 mM stock solution in dH2O), and 2
µL of deionized water. The click reaction mixture was gently
shaken for 2 hrs at 30 °C. The reaction was quenched with the
addition of 15 µL of 4x Laemmli sample buffer and the result-
ing sample was vortexed and centrifuged. Appropriate concen-
trations of labeled and clicked human cathepsin L were loaded
in 4-20% gradient SDS-PAGE gel (Bio-Rad Inc.) and the pro-
tein was resolved from other components. For control experi-
ments, identical labeling and click chemistry protocols were
utilized except that (i) heat-denatured and inactivated human
cathepsin L was utilized, and (ii) the labeling with KDP-1 was
performed in presence of the highly potent competitive inhibi-
tor KD-1 (net 15 µM).
4.5.3 Wound Healing Assay for Assessment of the
Effect of probe KDP-1 on Cell Migration: Approximately 3
5
x 10 MDA MB–231 cells were added to 24– well culture
plates containing 1 ml of complete medium and allowed to
attach and spread to confluency for 24 hours. A 200μl
Pipetteman tip was then used to wound the monolayer and
form a cell–free area. Any detached cells and cell debris
were removed by washing the well with complete medium
and then 1 ml of complete medium was added to the well.
Next, 1.25 µM or 3.75 µM KDP–1 or 0.5% DMSO as add-
4.5. Cell Biology Studies
4.5.1 Biocompatibility Assay: MDA MB–231
(ATCC ® HTB–26 ™) human breast cancer cells were plated
in 96-well tissue culture plates at 0.5 x 104 cells per well in
100μl complete medium (DMEM supplemented with 10%
FCS and 10% penicillin/streptomycin, nonessential amino

The manuscript was written through valuable contributions of all
authors. All authors have given approval to the final version of the
manuscript.
ed to the wells. Phase contrast photographs of the scratched
area were taken as a time zero reference for cell position.
The cells were returned to 37°C for 21 hours and then pho-
tographs were taken to determine how far the cells had
moved into the scratch. Images from the two time points
were overlaid and the distance between the initial and final
front were measured at 6 points along the fronts to deter-
mine distance of migration. The data represent the average
of four independent trials.
Funding Sources
S.K.P. gratefully acknowledges the financial support from the
National Science Foundation (NSF); Grant no. 1709711. K.R.F.
also thanks PSC–CUNY (Awards # 69356-00 47 and 61414-00
49). J.G. and K.R.F. thank NIH T 34 GM070387.
4.6 In Vivo Zebra Fish Hatching Experiment
ACKNOWLEDGMENT
Zebrafish embryos were a gift from the laboratory of
Dr. Nathalia Glickman Holtzman, Queens College. Approxi-
mately 18-20 embryos were place in 6–well plates containing
1 ml of embryo water containing 0.5% DMSO. Subsequently,
0.75 or 1.54 µM KDP–1 or 10 µM of the pan cysteine
cathepsin inhibitor E64 were added. The embryos were incu-
bated at 28.5°C and the number hatching from the chorion
(egg envelope) at various timepoints were then counted under
a dissecting scope.
The authors wish to thank Dr. Nathalia Glickman Holtzman,
Queens College for providing zebrafish embryos. This material is
based upon work supported by the National Science Foundation
(NSF) under Grant No. 1709711.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
publication’s website.
AUTHOR INFORMATION
Corresponding Author
*Correspondence: Sanjai.Kumar@qc.cuny.edu; Phone: +1
718 997 4120; Fax: +1 718 997 5531.
Author Contributions

Figure 1. Chemical structures of the representative small molecule probes developed for cysteine cathepsins.
Red: fluorescence reporter group; Pink: quencher; Blue: hydrolyzing amide bond; Green: photoactivatable
group.
8

Figure 2. Chemical structures of (a) the highly potent and selec-
tive inhibitory compound KD-1 that inhibits human cathepsin L
activity in a covalent and irreversible manner, and (b) the devel-
oped clickable and tagless activity-based human cathepsin L
probe, KDP-1
9

Scheme 1. Synthesis scheme utilized for the development of clickable and tagless
activity-based human cathepsin L probe, KDP-1; Reagents and conditions: (a)
triphosgene, Na2CO3, toluene, rt; (b) L-phenylalanine methyl ester hydrochloride,
N,N-diisopropylethylamine, CH2Cl2, rt; (c) lithium hydroxide, THF/water; (d) O-
(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HCTU), N,N-diisopropylethylamine, DMF, 0 °C(2 hr) -12 hr (rt); (e) n-
tetrabutylammonium iodide, acetone, reflux (f) triphenylphosphine, sulfuryl chloride
in CH2Cl2, 0 °C, rt; (g) 4-bromophenol, triethylamine, CH2Cl2, 0 °C, rt
10

[A]
[B]
Figure 3. Kinetic evaluation of cathepsin L probe, KDP-1: [A] Demonstration of time-dependent
inhibition of human cathepsin L activity by KDP-1 probe in a dose dependent manner. The pro-
gress curves is acquired by monitoring the hydrolysis of Z-FR-AMC substrate in presence of vary-
ing concentration of KDP-1 probe and control (DMSO). [B] Selectivity profile of KDP-1 probe
against a panel of cathepsins, a low pKa containing Cys enzyme, protein tyrosine phosphatase 1B,
and a serine protease, trypsin. NR = No reactivity at 50 µM KDP-1.
11

[B]
Human Cathepsin L Labeling
Control Experiments
[A]
a
100 ng
b
50 ng
20 ng
25 kDa
25 kDa
25 kDa
25 kDa
10 ng
5 ng
1 ng
Figure 4. Imaging of active human cathepsin L, labeled with ctABP, KDP-1. [A]: Pu-
rified and activated recombinant human cathepsin L was incubated with KDP-1 probe.
After reaction was quenched, the labeled enzyme was clicked with 5-TAMRA-azide,
resolved using SDS-PAGE, and detected using Typhoon 9400 scanner (ex: 532 nm,
em: 580 nm). The experiment indicates that as little as 1 ng (40 fmol) of active human
cathepsin L can be detected. [B] Control experiments: (a) heat-denatured inactivated
human cathepsin L was subjected to identical labeling and click chemistry procedures;
no labeling was observed (50 ng loaded), indicating KDP-1 is an activity-based probe.
(b) Identical labeling and detection procedures were performed as in [A], but in pres-
ence of a competitive cathepsin L inhibitor KD-1. Significantly reduced amount of
human cathepsin L labeling was observed, as expected. The bottom panel is the silver-
stained image of the same fluorescence gel. A representative of three independent ex-
periments is shown.
Mean ± s.d.
120%
Cellular Human Cathepsin L Activity
p < 0.05 compared to
100%
DMSO control
Figure 5. Assessment of KDP-1 probe’s biocompatibility and its ability to inhibit human
80%
cathepsin L activity in human MDA-MB-231 breast cancer cell lines. [A] Live MDA MB–
231 cells were treated (24 hrs) with KDP–1 (1-10 µM) and control (DMSO). Cell viability
60%
was assessed using colorimetric CytoScan WST–1 cell proliferation assay in a microplate
reader. No apparent cellular toxicity is observed, even at a concentration as high as 10 µM.
[B] Live MDA MB–231 cells were treated (24 hrs) with appropriate concentration of
40%
DMSO (-ve control), cathepsin L probe KDP-1, cell permeable cathepsin L inhibitor KD-1
(+ve control), and a pan cell permeable cathepsin L inhibitor E-64d (+ve control). After
wash, cells were permeabilized with digitonin, and cathepsin activities was measured using
20%
[A]
fluorescence-based cathepsin substrate, Z-FR-AMC. Dose-dependent loss of cathepsin ac-
tivities indicates that KDP-1 is cell penetrable, and is thus suitable for activity-based label-
12
0%
ing of human cathepsin L at the proteome levels.
0.5% DMSO
10 M KDP-1
750 nM KDP-1
10 M KD-1
10 M E-64d

Cell Viability (Relative to DMSO Control)
120%
100%
80%
60%
40%
20%
0%
10 M KDP-1
1.5 M KDP-1
5 M KDP-1
1 M KDP-1
0.5% DMSO
[A]
[B]
Cell front t = 0, Cell front t = 21 hrs
14
12
10
8
6
4
2
DMSO
(Control)
1.25 M
KDP-1
3.75 M
KDP-1
13

Figure 6. Phenotypic evaluation of cathepsin L probe,
KDP-1 in human MDA-MB-231 breast cancer cells. [A]
Wound-healing assay for assessing cell migratory poten-
tial where living cells grown to 95% confluency were
treated with control (DMSO) and KDP-1. A wound
(green borders, t = 0 hr) was created and the filling of the
wound (cell front) was assessed after 21 hrs (red borders).
Migratory potential of the cells are reduced when treated
with KDP-1, consistent with cathepsin L physiological
function. [B] Quantitative assessment of the cell migra-
tion rate when cells are treated with KDP-1.
[I]
[II]
0.5% DMSO
14

Figure 7: Inhibition of zebrafish embryo hatching from 29–
72 hours post fertilization by cathepsin L probe, KDP–1 and a
pan cysteine cathepsin inhibitor, E–64. [I] Phase contrast mi-
crographs of zebrafish embryos 53 hours post hatching and 29
hrs post treatment with (A) Control (0.6% DMSO), (B)
1.5µM KDP–1, and 10 µM E–64, a pan cysteine cathepsin
inhibitor. The white arrow indicates an empty chorion and the
black arrow indicates a hatched embryo. The asterisk indi-
cates an embryo still in the chorion. [II] A group of embryos
were exposed to probe KDP-1 (750 nM, and 1.5 µM), and E-
64 (10 µM) post-fertilization, and the number of embryos
hatched were counted and plotted. It is evident that hatching
is delayed when embryos are treated with inhibitory probe
KDP-1 (29-51 hpf), compared to control (0.6% DMSO). A
high concentration of pan cysteine cathepsin inhibitor E64
(10 µM) delays the hatching process significantly (only about
50% hatched), even at 54-72 hpf, and serves as a positive
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20

Cell Penetrable, Clickable and Tagless Activity-based Probe of
Human Cathepsin L
Dibyendu Dana,1,2 Jeremy Garcia,3 Ashif I. Bhuiyan1,6, Pratikkumar Rathod,4,7 Laura Joo,1 Daniel A.
Novoa,1 Suneeta Paroly,5 Karl R. Fath,3,6 Emmanuel J. Chang,4,2,6 Sanjai K. Pathak1,2,6,*
1Queens College of The City University of New York, Chemistry and Biochemistry Department, 65-30 Kissena Blvd, Flush-
ing, NY 11367, United States; 2Chemistry Doctoral Program, The Graduate Center of The City University of New York, 365
5th Ave, New York, NY, 10016, USA.; 3Queens College of The City University of New York, Department of Biology, 65-30
4
Kissena Blvd, Flushing, NY 11367, USA.; York College of The City University of New York, Department of Chemistry,
5
94-20 Guy R. Brewer Blvd, Jamaica, NY, 11451-0001, USA.; Bard High School Early College Queens, 30-20 Thomson
6
Avenue, Long Island City, USA; Biochemistry Doctoral Program, The Graduate Center of The City University of New
York, 365 5th Ave, New York, NY, 10016, USA.; 7Laguardia Community College, 31-10 Thomson Ave, Long Island City,
NY 11101, USA
Activity-based Functional Profiling of Human Cathepsin L Activities from Distinct Proteomic Origins
21

Highlights:
#
A highly sensitive and clickable activity-based probe of human cathepsin L, KDP-1, was devel-
oped.
#
#
#
Probe KDP-1 was selective and non-toxic to human cells.
Cellular and in-vivo studies demonstrated that KDP-1 probe was suitable for functional studies.
KDP-1 probe could find numerous applications in functional proteomics and cathepsin L-
targeted drug discovery endeavors.
22