Dual-Mechanism Quenched Fluorogenic Probe Provides Selective and Rapid Detection of Cathepsin L Activity**

Article abstract of DOI:10.1002/cmdc.202000823

Cathepsin L (CTL) is a cysteine protease demonstrating upregulated activity in many disease states. Overlapping substrate specificity makes selective detection of CTL activity difficult to parse from that of its close homologue CTV and the ubiquitous CTB. Current probes of CTL activity have limited applications due to either poor contrast or extra assay steps required to achieve selectivity. We have developed a fluorogenic probe, CTLAP, that displays good selectivity for CTL over CTB and CTV while exhibiting low background fluorescence attributed to dual quenching mechanisms. CTLAP achieves optimum CTL selectivity in the first 10 min of incubation, thus suggesting that it is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

Full text of DOI:10.1002/cmdc.202000823

Accepted Article
Title: Intrinsically quenched fluorogenic probe provides selective and
rapid detection of cathepsin L activity
Authors: Kelton A. Schleyer, Ben Fetrow, Peter Zannes Fatland, Jun
Liu, Maya Chaaban, Biwu Ma, and Lina Cui
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
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To be cited as: ChemMedChem 10.1002/cmdc.202000823
Link to VoR: https://doi.org/10.1002/cmdc.202000823
01/2020

10.1002/cmdc.202000823
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COMMUNICATION
Dual-mechanism quenched fluorogenic probe provides selective and rapid
detection of cathepsin L activity
Kelton A. Schleyer[a,b], Ben Fetrow[b], Peter Zannes Fatland[b], Jun Liu[a,b], Maya Chaaban[c], Biwu Ma[c],
and Lina Cui*[a,b]
[a]
K. A. Schleyer, Dr. J. Liu, Prof. L. Cui
Department of Medicinal Chemistry
UF Health Science Center, UF Health Cancer Center
University of Florida
1345 Center Dr., Gainesville, FL 32610 (USA)
E-mail: linacui@cop.ufl.edu
[b]
[c]
K. A. Schleyer, B. Fetrow, P. Z. Fatland, Dr. J. Liu, Prof. L. Cui
Department of Chemistry and Chemical Biology
UNM Comprehensive Cancer Center, University of New Mexico
300 Terrace St. NE, Albuquerque, NM 87131 (USA)
M. Chaaban, Prof. B. Ma
Department of Chemistry and Biochemistry
Florida State University
95 Chieftan Way 118 DLC, Tallahassee, FL 32306 (USA)
Supporting information for this article is given via a link at the end of the document.
from that of its close homologue CTV, as such closely related
cathepsins exhibit markedly different activity against certain
endogenous substrates.[20]
Abstract: Cathepsin L (CTL) is a cysteine protease demonstrating
upregulated activity in many disease states. Overlapping substrate
specificity makes selective detection of CTL activity difficult to parse
from its close homologue CTV and the ubiquitous CTB. Current
probes of CTL activity have limited applications due to either poor
contrast or extra assay steps required to achieve selectivity. We have
developed
a fluorogenic probe, CTLAP, which displays good
selectivity for CTL over CTB and CTV while exhibiting low background
fluorescence attributed to dual quenching mechanisms. CTLAP
achieves optimum CTL selectivity in the first 10 min of incubation
suggesting that it is amenable for rapid detection of CTL, even in the
presence of competing cathepsins.
The cathepsin family of lysosomal proteases includes 11
members associated with various pathological conditions[1]
including cancer[2]. Although their redundancy in certain contexts
has been described,[3] they exhibit distinct tissue distribution[4] and
singular cathepsins serve as useful biomarkers for cancers[5] or
other diseases.[6] Among many roles, CTL is involved in cancer
progression by directly degrading the extracellular matrix (ECM)
components[7] (including laminin[8] and some forms of collagen[9])
while also activating other ECM-degrading enzymes, such as
Figure 1. A) CTLAP has intrinsically quenched emission, leading to high
contrast. leading to selective detection of CTL over related cathepsins (CTB,
CTV, CTS). B) Alternative designs of fluorescent probes show reduced
selectivity or contrast.
The development of selective cathepsin probes is challenging
due to overlapping substrate selectivity among cathepsins.
Detection of CTL activity commonly uses simple fluorogenic
substrates such as Z-FR-AMC, however this probe exhibits off-
target activation by CTB and other proteases.[21] This type of
probe, in which the fluorophore is masked by a substrate
sequence or quenching group, can provide high contrast when
such groups are removed by the target enzyme.[22] However, its
low selectivity often requires inclusion of an exogenous
inhibitor[5a, 6b, 12, 23] or pre-incubation under harsh conditions (4 M
urea for 30 min)[24] to deactivate competing enzymes (namely
CTB) to achieve selectivity for CTL. A probe with greater
selectivity for CTL could eliminate the need for such steps,
enabling less intrusive and more convenient real-time detection
of CTL activity. These concerns are mitigated with highly
selective probes, which have been discovered for CTL activity
by screening combinatorial substrate libraries,[14c, 25] typically
requiring generation and testing of thousands to hundreds of
thousands of compounds.[26] While highly selective, these
probes often lack fluorescence quenching mechanisms[22a-c] and
therefore provide bright labeling but poor contrast until inactive
10]
heparanase, to promote an aggressive phenotype.[4d,
Cathepsin L levels in serum and urine have been shown to
increase in cancer patients compared to healthy patients and tend
to correlate with tumor grade, invasive potential, and metastatic
spread.[11] Likewise, CTL contributes to atherosclerosis by
degrading the medial elastica laminae and enabling smooth
muscle cell and leukocyte migration into atherosclerotic
plaques,[12] and serum CTL levels correlate with the presence of
coronary artery stenosis.[13]
Since enzymatic activity does not always correlate with
expression at the protein[14] or mRNA level,[15] it is equally critical
to detect the enzymatic activity of CTL.[16] However, detection of
a single cathepsin is necessary to parse relative contribution in
contexts involving multiple cathepsins. For instance, CTB, CTL,
and CTS are simultaneously involved in atherosclerosis,[17] but
each contributes to the process through different mechanisms.[12]
It is also crucial in contexts where specific cathepsins may have
opposing effects; for example, in some contexts CTL deficiency
can be tumorigenic,[18] a correlation believed to be unique to this
cathepsin.[19] Activity of CTL should be considered distinct even
1
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probes are washed out of the sample,[17, 25a, 27] compromising
their use in rapid and single-step detection.
Figure 3. HPLC traces of CTL activation of CTLAP and Z-FR-AMC to
generate AMC. Detection wavelength = 325 nm.
We examined the spectral characteristics of CTLAP (Figure
2). The absorption spectrum (Figure 2, solid) exhibits three local
maxima at 298 nm, 316 nm, and 330 nm. The 330 nm peak is
attributed to the attached AMC group, consistent with the reported
λmax value for 7-amido-4-methylcoumarin.[33] The peak at 298 nm
is attributed to the peptide backbone, with its greater prominence
reasonably achieved if the absorbance curves of Z-FR-AMC and
2 are summed. The peak at 316 nm is likely also the result of
spectral overlap between these two portions of the probe. The
emission curve (Figure 2, dotted) has a maximum around 390 nm,
again consistent with literature.[33] The excitation curve at 445 nm
emission (Figure 2, dashed) mirrors the absorbance curve almost
identically; this suggests that all portions of the molecule
contributing to the absorption in this range will also contribute to
emission. The excitation spectrum of masked AMC alone (in the
form of Z-FR-AMC) lacks the prominent band at 290 nm, while
the excitation of compound 2 under identical conditions shows no
emission. This suggests that higher-energy absorbance by 2-
phenylthiophene can also contribute to masked AMC emission,
although this is not likely under the conditions typically used for
AMC-based fluorescence assays (excitation around 350 nm). We
next tested whether cathepsins could activate CTLAP and
release the AMC reporter (Figure 3). Incubation with cathepsins
resulted in consumption of CTLAP and generation of a new peak
with retention time of 10.7 min, consistent with that of AMC, while
the emission spectrum shifted to a maximum of 450 nm, also
consistent with the generation of AMC (Figure S1).
We investigated whether CTLAP would retain selectivity for
CTL against the most similar competing cathepsins: CTB, CTV,
and CTS (Figure 2). These cathepsins were selected based on
their similarity to CTL in substrate specificity or in their common
presence in applications of CTL detection. CTV is a close
homologue of CTL (being initially characterized as “cathepsin
L2”[4b]) and the challenge of its remarkably overlapping substrate
specificity is mitigated only by its isolated expression to a few
tissues. CTB is the most notable competitor of CTL, being the
most highly and ubiquitously expressed cathepsin.[4d] CTS was
also examined due to its organization close to CTL in sequence
homology.[25d] We incubated CTLAP with each cathepsin and
monitored the increase in fluorescence emission over time to
determine selectivity. Activation of CTLAP by CTL completed the
initial velocity phase within the first 10 min, followed by formation
of a plateau (Figure S2). In contrast, the other cathepsins
remained in their initial velocity phase throughout the first 1 h of
the assay. The selectivity for CTL was determined from the
relative signal intensity generated by each cathepsin, compared
to that from CTL (Figure 4A,B). CTLAP generated low off-target
signal from each competing cathepsin tested: after the first 10 min
Figure 2. Structures of CTLAP, Z-FR-AMC, and compound 2, along with their
absorption (solid line), emission, (large dot), and excitation (small dot) spectra.
Emission spectra collected with excitation at 350 nm. Excitation spectra
collected with emission at 445 nm.
We report a cathepsin-L activable probe (CTLAP) that
experiences dual quenching[28] by its own substrate structure,
precluding the need to incorporate exclusive quenching groups in
order to enhance emission turn-on (Figure 1).[29] The probe
exhibits low background signal and over 120-fold turn-on ratio
while demonstrating high selectivity for CTL over competing
cathepsins within the first 10 min of incubation. These attractive
characteristics bridge the gap in current CTL probes, providing a
combination of high selectivity and high contrast.
To design CTLAP, we selected inhibitor scaffolds providing
selectivity for CTL over multiple other cathepsins.[29a, 30] Selectivity
against multiple cathepsins is desirable because it increases the
potential applications of a CTL probe, as each pathological model
will exhibit a unique set of competing cathepsins.[7c, 31] In seeking
attractive inhibitor scaffolds, one notable result was a 2-
phenylthiophene residue that provided selectivity for CTL over
multiple related cathepsins[30b]
. While examination of the
cathepsin binding pockets elicits no obvious explanation for the
selectivity provided by this residue, it appears to be a privileged
CTL-selective scaffold even among extended aromatic groups.[29g,
30b] We therefore constructed CTLAP (Figure 2) by incorporating
the 7-amino-4-methylcoumarin (AMC) fluorophore with the
peptide sequence containing this 2-phenylthiophene residue.
Inclusion of a labile linker between the fluorophore and inhibitor
scaffold was avoided, as such linkers have been shown to shift
selectivity away from CTL and toward CTB.[32] The probe was
accessed by solution-phase peptide synthesis using an
Fmoc/Boc strategy (see Supplementary Information and Scheme
S1). The novel compound
2 was synthesized from 4-
iodophenylalanine by N-carboxybenzylation followed by Suzuki
coupling with thiophene-2-boronic acid. Intermediate 2 underwent
amide coupling with H-Lys(Boc)-AMC and subsequent Boc
deprotection afforded CTLAP.
2
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Figure 4. Selectivity of CTLAP and Z-FR-AMC for cathepsins. Relative signal intensity generated by cathepsins with (A) CTLAP and (B) Z-FR-AMC at pH 6.5.
Horizontal red line marks 0.2 (20%) relative signal. (C) Turn-on ratio of probes within 10 min, pH 6.5.
of incubation, the ubiquitous CTB generated only 6% relative
signal, while the highly homologous CTV generated only 15%
relative signal (Figure 4A, Table S1). This translates to a
selectivity profile of 20-fold for CTB and 6-fold for CTV. When Z-
FR-AMC was used, CTB generated at least 20% relative signal
during this time window, while CTV maintained at least 16% signal
(Figure 4B, Table S1), giving a selectivity profile of 6-fold for CTV
but only 5-fold against CTB, the more common competitor. This
suggests CTLAP is more suitable for applications similar to those
of Z-FR-AMC, including determination of CTL activity in clinical
samples.[5a, 6b] While this selectivity profile is eclipsed by highly
selective CTL probes,[34] few of these contain fluorescence
contrast mechanisms, again revealing that CTLAP strikes a
balance between favorable CTL selectivity and detection contrast.
Cathepsins are typically inactive at pH 7.4 but active in a
broad range of acidic pH,[4c, 29b, 35] consistent with their localization
within lysosomes and sustained activity in the acidic extracellular
space in tumors. Therefore, we tested the response of CTLAP to
cathepsin activity at pH 5.0 and 6.5, reproducing common assay
conditions for cathepsin activity while probing for possible
differences in activity under these conditions (Figure S2). CTL
exhibited faster turnover of CTLAP at pH 6.5 than at pH 5.0,
suggesting 6.5 to be the optimal pH for detection with CTLAP
(Figure S2A,C). Significantly, CTLAP also demonstrated greater
selectivity for CTL at pH 6.5, as CTB activity was greatly reduced
at this pH value. These results support the use of pH 6.5 for
optimal selectivity with CTLAP.
emission processes of CTLAP. The absorbance of CTLAP is
linear within the concentration range used (Figure S5), and the 2-
phenylthiophene residue does not absorb at this wavelength
(Figure 2). We thus hypothesized that CTLAP emission was
attenuated somehow, resulting in reduced background emission
(Figure S4B). The emission of some fluorophores is altered by
assay pH, often due to the existence of a non- or low-emissive
conjugate acid or base form of the fluorophore,[36] but no such
effect was observed for AMC (Figure S6).
While exploring the causes of the low background
fluorescence of CTLAP, we considered the possibility that the
unique 2-phenylthiophene residue of CTLAP was quenching the
attached AMC.[37] To examine this, we performed density
functional-theory (DFT) calculations to determine the relative
HOMO and LUMO energy levels of AMC and the 2-
phenylthiophene moiety of CTLAP (Figure 5). Quenching of AMC
emission by benzene or 2-phenylthiophene through energy
transfer is unlikely, because the bandgap of AMC is smaller than
that of the other moieties. Electron transfer from AMC to benzene
in Z-FR-AMC is unlikely because of the high LUMO and deep
HOMO of benzene. In CTLAP, electron transfer (hole transfer)
between AMC and 2-phenylthiophene might be possible due to
the comparable HOMO levels, which could quench the emission
of AMC. To test this hypothesis, the quantum yield and molar
attenuation coefficient of CTLAP were measured (Table 1, Figure
S7). CTLAP has a higher molar attenuation coefficient than Z-FR-
AMC but a markedly lower quantum yield (Table 1), supporting
the hypothesis that AMC emission is reduced in CTLAP by
quenching mechanisms that are not present in Z-FR-AMC. This
hole transfer mechanism likely does not influence the emission of
the probe after activation by CTL, as the enzymatic reaction
cleaves the fluorophore from the substrate and allows diffusion
outside of the range of quenching. Complementing this,
conversion to free AMC from the respective 7-acetamido-4-
methylcoumarin increases the quantum yield nearly four-fold.[33]
Strikingly, CTLAP maintained a significantly greater turn-on
ratio with CTL compared to other cathepsins across the entire
assay (Figure S3), most notably within the first 10 min of
incubation (Figure 4C, Table S1). After 24 h incubation with
cathepsins, CTLAP exhibits a final turn-on ratio greater than 120-
fold, likely due to its low background fluorescence (Figure S4),
resulting in much greater contrast upon activation by CTL. This
could possibly be due to interference in either the absorption or
3
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consideration other possible dual-purpose structural components.
Our work reveals that while inhibitor-based probe design can
retain the selectivity of the parent inhibitor, it can also result in the
discovery of emergent properties that further improve the nature
of the probe created. Current work is examining clinical
applications of CTLAP that take advantage of these attractive
characteristics.
Thus, the 120-fold turn-on ratio of CTLAP is attributed to the
compounding effect of two quenching mechanisms, the first being
a standard off-on mechanism caused by release of AMC[21] while
the second is a unique quenching event caused by the 2-
phenylthiophene residue that also endows the probe with high
selectivity for CTL over competing cathepsins. This could be
linked to the effect of adding a quenching dye to the probe to
reduce the background of always-on probes[26d, 38] (a common
strategy for CTL imaging probes) without the need to actually add
such a moiety. The idea of probe components (such as a self-
immolating aromatic[32] or aliphatic[25a] spacer, or reporter and
Acknowledgements
quencher location[39]
)
influencing selectivity has been
This work was supported by research grants to Prof. L. Cui from
the National Institute of General Medical Sciences of National
Institutes of Health (Maximizing Investigators' Research Award for
Early Stage Investigators, R35GM124963), and the Department
of Defense Career Development Award (W81XWH-17-1-0529).
We are grateful to the support from the University of New Mexico
(UNM), the UNM Comprehensive Cancer Center and the National
Cancer Institute of the United States (P30CA118100). NMR
spectra were collected in part from the NMR Facility, Department
of Chemistry and Chemical Biology, UNM, and from the
Department of Medicinal Chemistry, College of Pharmacy,
University of Florida (UF). Mass spectrometry services were
provided in part by the Mass Spectrometry Facility, Department of
Chemistry and Chemical Biology, UNM, and from the Mass
Spectrometry Research and Education Center, Department of
Chemistry, UF (NIH S10 OD021758-01A1).
demonstrated, but here we show the reverse, that probe
components included to increase selectivity can influence the
photophysical properties of the probe. This dual-purpose design
is attractive in theory as it reduces the molecular complexity of the
designed probe. To translate this to longer emission wavelengths
ideal for fluorescent biological probes, a different substrate moiety
with a HOMO-LUMO gap matching that of the new red-shifted
fluorophore must be identified which still provides a favorable
selectivity profile among cathepsins.
Keywords: cathepsin • enzymes • fluorescent probes •
medicinal chemistry • quenching
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Compound
ε x [10-3] [a, b]
ΦF
CTLAP
11.0
7.5
0.02
0.32
Z-FR-AMC
[a] Values standardized against methylumbelliferone (see Supporting
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10.1002/cmdc.202000823
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A novel cathepsin L activable probe (CTLAP) bears a chemical structure that simultaneously provides high selectivity for its target
while quenching its own background emission, resulting in superior contrast within 10 min of incubation time and 120-fold turn-on
fluorescence upon consumption. Along with being a novel detection tool, CTLAP reveals that chemical structure can influence
photophysical properties as well as the selectivity of such tools.
6
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