Prodrug-inspired probes selective to cathepsin B over other cysteine cathepsins

Article abstract of DOI:10.1021/jm500544p

Cathepsin B (CTB) is a cysteine protease believed to be an important therapeutic target or biomarker for several diseases including aggressive cancer, arthritis, and parasitic infections. The development of probes capable of assessing CTB activity in cell lysates, living cells, and animal models of disease are needed to understand its role in disease progression. However, discovering probes selective to cathepsin B over other cysteine cathepsins is a significant challenge due to overlap of preferred substrates and binding site homology in this family of proteases. Herein we report the synthesis and detailed evaluation of two prodrug-inspired fluorogenic peptides designed to be efficient and selective substrate-based probes for CTB. Through cell lysate and cell assays, a promising lead candidate was identified that is efficiently processed and has high specificity for CTB over other cysteine cathepsins. This work represents a key step toward the design of rapid release prodrugs or substrate-based molecular imaging probes specific to CTB.

Full text of DOI:10.1021/jm500544p

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Article
Prodrug-Inspired Probes Selective to
Cathepsin B Over Other Cysteine Cathepsins
Morshed Alum Chowdhury, Ignace Adolfo Moya, Shardul Bhilocha, Cody Curtis
McMillan, Brady Garitt Vigliarolo, Ingeborg Zehbe, and Christopher Peter Phenix
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jun 2014
Downloaded from http://pubs.acs.org on July 2, 2014
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ProdrugꢀInspired Probes Selective to Cathepsin B
Over Other Cysteine Cathepsins
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Morshed A. Chowdhury,ᴪ Ignace A. Moya,ᴪ Shardul Bhilocha,ǂ Cody C. McMillan,ǂ Brady G.
Vigliarolo,ǂ Ingeborg Zehbe, ᴪ, ǂ,◊ and Christopher P. Phenix,ᴪ, ǂ,◊,∗
ᴪThunder Bay Regional Research Institute, 2321ꢀ 290 Munro Street, Thunder Bay, Ontario, Canada,
P7A 7T1.
ǂDepartment of Biology and Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay,
Ontario, Canada, P7B 5E1
◊Northern Ontario School of Medicine, Medical Sciences Division, 955 Oliver Road, Thunder Bay,
Ontario, Canada, P7B 5E1
KEYWORDS: Cathepsin B, Fluorogenic Peptides, Enzyme Kinetics, Cell Lysate Assays, Prodrug
Linker, Live Cell Assays
ABSTRACT: Cathepsin B (CTB) is a cysteine protease believed to be an important therapeutic
target or biomarker for several diseases including aggressive cancer, arthritis and parasitic
infections. The development of probes capable of assessing CTB activity in cell lysates, living cells
and animal models of disease are needed to understand its role in disease progression. However,
discovering probes selective to Cathepsin B over other cysteine cathepsins is a significant challenge
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due to overlap of preferred substrates and binding site homology in this family of proteases.
Herein, we report the synthesis and detailed evaluation of two prodrug inspired fluorogenic peptides
designed to be efficient and selective substrateꢀbased probes for CTB. Through cell lysate and cell
assays, a promising lead candidate was identified that is efficiently processed and has high
specificity for CTB over other cysteine cathepsins. This work represents a key step towards the
design of rapid release prodrugs or substrateꢀbased molecular imaging probes specific to CTB.
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INTRODUCTION
The papain family of cysteine proteases includes 11 human enzymes (cathepsins B, C, F, H, K, L,
O, S, V, W and X) responsible for lysosomal protein degradation in many biological and
pathological processes.1 Cysteine cathepsins are synthesized as inactive “proenzymes” whose
expression is controlled by transcriptional and translational mechanisms. The regulation of enzyme
activity is accomplished through a variety of posttranslational processes, the most important being
the protease activation of the proenzyme in the acidic environment of the endosomes/lysosomes and
the expression of high affinity endogenous inhibitors from the cystatin family.2 Significant
increases in functional enzyme can result from alterations in posttranslational mechanisms3,4
highlighting the need for techniques that reveal enzyme activity when evaluating cysteine proteases
in disease promoting processes.
Cathepsin B (CTB, EC 3.4.22.1) is a promising diagnostic and prognostic biomarker of various
cancers5ꢀ9 and is a member of a tumor promoting proteolytic network.10ꢀ12 CTB appears to affect
cancer progression based upon its localization with aggressive cancers having high CTB activity at
the invading edge of a tumor and in the extracellular matrix secreted by a variety of cell types.13 In
contrast, intracellular CTB appears to inhibit cancer by enhancing apoptosis.14 Recent reports
suggest CTB, together with the related enzyme cathepsin L (CTL, EC 3.4.22.15), may protect
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against anticancer chemotherapies such as Taxol, meaning CTB and/or CTL activity may be a
useful marker predictive of response to chemotherapy.15 Sensitive and specific tools capable of
assessing CTB activity in cell lysates, cell culture and animal models of aggressive cancer are
needed to validate this enzyme as a high priority cancer marker or therapeutic target.
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To provide information beyond protease expression and to instead directly evaluate CTB activity
in complex mixtures, cells, and animals, two strategies have recently emerged. The first was
developed by Bogyo and coworkers16ꢀ18 who designed elegant activityꢀbased probes that efficiently
label CTB and CTL with a fluorescent tag. These optical probes are irreversible inhibitors that label
active enzyme and are wellꢀsuited for detecting and localizing CTB and CTL activity in vitro and in
vivo. Although specificity towards CTB over CTL was never achieved in the optical agents,
derivatives of these compounds were radiolabeled for positron emission tomography (PET) studies
to evaluate cysteine protease activity in animal models of cancer.19 While modest tumour uptake
was observed in the PET images, a high percentage of the injected dose was found in the liver and
eliminated through hepatobiliary clearance. Indeed, some of the most wellꢀestablished and
promising PET probes for imaging enzyme activity in cancer are small molecule substrates that
accumulate in the tumor and are rapidly cleared through the renal system.
The second strategy seeks to improve cellular and in vivo sensitivity by exploiting the catalytic
power of CTB since a single active protease can amplify the fluorescent signal by continuous
turnover of many substrateꢀbased probes. To evaluate this approach, a synthetic graft copolymer
consisting of polyꢀ(S)ꢀlysine modified with a protease releasable Cy5.5 nearꢀinfrared dye was
assessed in cancer cells and tumor xenograft mice.20 Although the polymer sensitively revealed
protease activity in vivo, low specificity towards CTB was observed since serine proteases also
activated this probe. Kim and coꢀworkers21 extended this strategy by developing a nanoꢀsized
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polymer decorated with peptides conjugated to a nearꢀinfrared fluorescent dye and a quencher
molecule that is released upon CTB hydrolysis. Although strong fluorescence was observed in
cancer cells and tumors that correlated with CTB expression, a polymer approach is largely
dependent upon the enhanced permeability and retention (EPR) effect for efficient tumour delivery.
As a consequence, polymer uptake and imaging performance can vary because the EPR effect is
heavily influenced by the conditions of tumor growth in xenografts and the specific cancer cell lines
used.22 Although substrateꢀbased polymers may offer higher sensitivity over the activityꢀbased
inhibitors, small molecule probes are easier to prepare, purify and usually have pharmacodynamic
properties superior for clinically established molecular imaging techniques like PET.
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Despite the potential benefits that small molecule substrates have over the activityꢀ and polymerꢀ
based probes for molecular imaging, designing compounds specific to CTB over other lysosomal
cysteine cathepsins is an enormous challenge since the S1, S1’ and S2’ recognition sites of these
enzymes are almost identical.23 Therefore, it is advantageous to thoroughly assess new compounds
for CTB efficiency and specificity over the other cysteine cathepsins early on in the discovery
process and prior to in vivo studies. A standard protocol used to evaluate the specificity of substrateꢀ
based fluorogenic probes involves the incubation of each probe candidate with a series of enzymes
having similar substrate preference or related family members. A graph of fluorescence activation
vs time is plotted to compare the relative rates of probe turnover by each protease as a qualitative
measure of specificity. Unfortunately, these experiments can be misleading especially when
saturating levels of probe are used which do not reflect intracellular or in vivo conditions where
probe concentrations are expected to be much lower. In contrast, the MichaelisꢀMenton parameters
KM (defined as the concentration of substrate at which the initial velocity is oneꢀhalf the maximal
velocity) and kcat (defined as the catalytic constant for the conversion of substrate to product)
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provide detailed insight into the recognition and catalytic efficiency of a substrate or probe
candidate towards the target enzyme. The ratio of these two parameters, the “specificity constant”
(kcat/KM), in general, reflects enzymatic efficiency at low substrate concentrations by combining
both affinity to the enzyme (KM) and catalytic turnover (kcat).24 Values of kcat/KM may better predict
probe performance inside living cells where the concentration of substrate is likely below the KM
value, meaning enzyme reaction rates are a second order process proportional to kcat/KM values. In
addition, kcat/KM is useful to compare probe turnover by related proteases and to identify lead
compounds as viable candidates for in vivo imaging using benchmark kinetic values established by
PET tracers that image enzyme activity in humans.25, 26
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To determine if CTB is a legitimate biomarker and/or therapeutic target of aggressive cancer,
small molecule PET probes that are efficient and specific substrates of CTB would be a valuable
tool for imaging studies on animal models of cancer. However, small molecule substrates that are
cell permeable and selective to CTB have not been reported to date. In this study, we have
synthesized and evaluated twoꢀcomponent (substrateꢀfluorophore) and prodrug inspired threeꢀ
component (substrateꢀlinkerꢀfluorophore) fluorogenic peptides that release 7ꢀaminoꢀ4ꢀ
methylcoumarin (AMC) upon protease hydrolysis. Using an in vitro screening platform that
includes kinetic studies, lysate and cell assays, we have identified one candidate that is simple to
prepare, biocompatible, cell permeable and may be useful in the design of rapid release prodrugs or
substrateꢀbased PET molecular imaging probes specific to CTB.
RESULTS AND DISCUSSION
Probe Design. As a key step in identifying efficientlyꢀprocessed CTB substrates that could be
later adapted into probes useful for sensitive fluorescence microscopy or PET molecular imaging,
we sought smallꢀmolecule lead compounds which are easily synthesized, fluorogenic, and specific
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to CTB over other cysteine cathepsins. To develop an experimental platform for identifying lead
candidates that meet the above requirements, we adapted the chemistry developed for activatable
“profluorophore” probes based on a threeꢀcomponent, prodrugꢀinspired strategy. Imaging probes of
this design consist of: a peptide portion that provides high affinity and specificity to CTB, a selfꢀ
destructive linker that spontaneously releases a reporter upon its enzymatic removal of the peptide
and a latent fluorophore which becomes highly fluorescent once enzymatically freed from the intact
probe (Figure 1). The prodrug linker pꢀaminobenzyl alcohol (PABA), originally reported by
Katzenellenbogan,27 was chosen because it can be conveniently coupled to peptides through its
amino group, allowing stable conjugation of alcohol and anilineꢀbased fluorophores and drugs.28-32
As the fluorescent reporter molecule, we chose AMC which is often used in the synthesis of
fluorogenic peptides to evaluate the substrate specificities of cysteine cathepsins. In addition, AMC
would be useful to evaluate probe specificity in cell lysate and preliminary fluorescence microscope
assays.
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Figure 1: A prodrug inspired strategy for sensing CTB activity. To identify substrates that are
efficient and selective substrates of CTB, we employed AMC as a latent fluorophore that becomes
fluorescent once released from the intact probe.
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It is well established that most human cysteine cathepsins prefer a hydrophobic amino acid at P2
and a positively charged substituent at the P1 positions.33 Therefore, most twoꢀcomponent
benzyloxycarbonyl (Cbz) protected substrates like CbzꢀPheꢀArgꢀAMC cannot be used to analyze
the activity of a specific cysteine cathepsin in samples having mixtures of these enzymes such as
cell lysates or living cells. Although CbzꢀArgꢀArgꢀAMC is the substrate of choice for assaying CTB
activity in cell lysates under acidic and reducing conditions, it is not commonly used in cell assays
presumably due to poor cell membrane permeability. The recognition of doubly cationic substrates
such as CbzꢀArgꢀArgꢀAMC by CTB has been attributed to a glutamic acid residue at the top of the
S2 binding pocket.34 Other related enzymes lack an equivalent glutamic acid residue and prefer
hydrophobic amino acids at the P2 position of substrates, explaining the selectivity of CbzꢀArgꢀ
ArgꢀAMC for CTB. We decided to synthesize CbzꢀPheꢀLysꢀAMC (1)35 and CbzꢀLysꢀLysꢀAMC (2)
to evaluate how substitution of Lysine for Arginine would affect recognition and turnover by CTB
and CTL and to serve as control substrates for the PABAꢀmodified compounds described below.
Using Lys instead of Arg is synthetically attractive given the convenience of installing and
removing the tertꢀbutoxycarbonyl (BOC) protecting group from the εꢀamine of (S)ꢀlysine.
Previous work has demonstrated that PABAꢀcontaining peptides modified into prodrugs are
serum stable and efficiently processed by CTB to release a cytotoxic drug.29,30 This suggests that
PABA and a bulky moiety like a drug or fluorophore are well tolerated at the P1' and P2' positions
respectively, and that a threeꢀcomponent prodrug inspired probe may be efficiently processed by
CTB. In addition, threeꢀcomponent peptides may offer higher specificity towards CTB because the
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probe extends into the core S3 ꢀ S2' regions (Figure 2a).23 To evaluate the efficiency of CTB to
activate the prodrugꢀinspired probes bearing the PABA spacer, we synthesized CbzꢀPheꢀLysꢀ
PABAꢀAMC (3) and CbzꢀLysꢀLysꢀPABAꢀAMC (4) (Figure 2b).
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Figure 2. (a) The recognition of the three component peptides by cysteine cathepsins. Cysteine
cathepsins bind to their substrates in an active site cleft where the wellꢀdefined S2, S1 and S1’sites
primarily determine substrate preference. Additional interactions between the substrate and the S3
and S2’ sites can influence recognition and must be considered when designing artificial substrates.
The three component probes have phenylalanine or lysine occupying the S2 site and lysine at the S1
site while Cbz, PABA and AMC extend into the S3, S1’ and S2' binding regions respectively. (b)
The fluorogenic peptides synthesized over the course of this work.
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Chemistry. The synthesis of the two component fluorogenic peptides was conducted using
routine solution peptide chemistry as shown in Scheme 1. The synthesis of CbzꢀPheꢀLysꢀAMC (1)
began by the coupling HꢀLysꢀNꢀεꢀBOCꢀAMC (5)36 with CbzꢀPheꢀOꢀsucinnimide (6) using sodium
bicarbonate in aqueous THF37 to produce CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀAMC (7). Subsequent
deprotection of the BOC group from 7 proceeded smoothly in 50% TFA/CH2Cl2 to yield 1. Using a
similar strategy, gramꢀscale amounts of CbzꢀLysꢀLysꢀAMC (2) were readily prepared by coupling 5
with CbzꢀLysꢀNꢀεꢀBOCꢀOꢀsuccinimide (8) in DMF in presence of triethyl amine to give CbzꢀLysꢀ
NꢀεꢀBOCꢀLysꢀNꢀεꢀBOCꢀAMC (9) followed by BOC deprotection in a 50% TFA/CH2Cl2 (Scheme
1).
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Scheme 1. Synthesis of the two component fluorogenic peptidesa
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Ph
O
H
O
a
N
H2N
CbzHN
N
H
O
O
N
H
O
O
O
O
O
CbzHN
N
O
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NHBoc
7 (81%)
O
NHBoc
CbzHN
5
Ph
6
O
c
O
N
Ph
CbzHN
O
O
O
H
b
N
O
O
N
H
O
NHBoc
8
NHBoc
NH2 . TFA
1 (quant.)
NH2 . TFA
O
H
N
CbzHN
N
H
O
O
O
O
c
H
N
CbzHN
N
H
O
O
O
9 (98%)
NHBoc
NH2 . TFA
2 (58%)
aReagents and conditions: (a) NaHCO3, THFꢀH2O, rt, 16 h; (b) Et3N, DMF, 0 °C to rt, 16 h; (c)
TFAꢀCH2Cl2 (1:1, v/v), iceꢀbath, 15 min.
As shown in Scheme 2, the synthesis of the three component fluorogenic peptide CbzꢀPheꢀLysꢀ
PABCꢀAMC (3) started with the coupling of HꢀLysꢀNꢀεꢀBOCꢀOH (10) with 6 to provide the
dipeptide CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀOH (11). Conjugation of the PABA linker with 11 was carried
out in presence of Nꢀethoxycarbonylꢀ2ꢀethoxyꢀ1,2ꢀdihydroquinoline (EEDQ)38 to give CbzꢀPheꢀ
LysꢀNꢀεꢀBOCꢀPABꢀOH (12). Efforts to prepare the AMC carbamate CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀ
PABCꢀAMC (14) failed when treating peptide 12 with pꢀnitrophenyl chloroformate,37 1,1'ꢀ
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carbonyldiimidazole,39 or phosgene, likely due to the low nucleophilicity of the nitrogen of AMC.
Efficient incorporation of AMC to yield carbamate 14 was ultimately accomplished following the
reaction of 12 with 7ꢀisocyanatoꢀ4ꢀmethylchromenꢀ2ꢀone (13) which was generated by refluxing of
AMC with phosgene. Removal of the BOC group from 14 with TFA/CH2Cl2 furnished the TFA salt
of probe CbzꢀPheꢀLysꢀPABCꢀAMC (3). To improve the aqueous solubility of probe 3, deprotection
of BOC from 14 with methanolicꢀHCl gave the HCl salt as shown in Scheme 2. Similarly, CbzꢀLysꢀ
NꢀεꢀBOCꢀLysꢀNꢀεꢀBOCꢀPABꢀOH (16) was prepared by conjugating the peptide CbzꢀLysꢀNꢀεꢀ
BOCꢀLysꢀNꢀεꢀBOCꢀOH (15) with PABA following the same procedure as used for the preparation
of 12. Coupling of 16 with isocyanate 13 afforded the carbamate CbzꢀLysꢀNꢀεꢀBOCꢀLysꢀNꢀεꢀBOCꢀ
PABCꢀAMC (17). The BOC deprotection from carbamate 17 with TFA/CH2Cl2 furnished the TFA
salt of CbzꢀLysꢀLysꢀPABCꢀAMC (4) (Scheme 3).
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Scheme 2. Synthesis of the three component fluorogenic peptides 3a
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7
H2N
OH
8
9
O
PABA
Ph
Ph
O
OH
H2N
O
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H
H
OH
6, a
N
N
CbzHN
N
H
CbzHN
OH
b
O
O
NHBoc
10
12 (94%)
11 (82%)
NHBoc
d
NHBoc
c
O
O
O
C
N
O
O
H2N
AMC
13
O
Ph
O
O
N
O
O
H
H
N
CbzHN
N
H
O
NHBoc
e or f
14 (38%)
O
Ph
O
O
N
H
O
O
H
N
CbzHN
N
H
O
NH2 . X
3.TFA; X = TFA (quant.)
3.HCl; X = HCl (87%)
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aReagents and conditions: (a) NaHCO3, THFꢀH2O, rt, 16 h; (b) THF, EEDQ, rt, 16 h; (c) 15%
phosgene in toluene, 120°C, 16 h; (d) THF, 80 °C, 2 h; (e) TFAꢀCH2Cl2 (1:1, v/v), iceꢀbath, 15 min;
(f) MethanolicꢀHCl (0.5M), rt, 16 h.
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Scheme 3. Synthesis of the three component fluorogenic peptide 4a
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NHBoc
4
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O
H
8, a
PABA, b
N
10
OH
CbzHN
O
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NHBoc
NHBoc
O
OH
13, c
H
N
N
CbzHN
H
O
16 (65%)
NHBoc
NHBoc
O
O
O
N
O
O
d
H
H
N
N
H
CbzHN
O
17 (83%) NHBoc
NH2 . TFA
O
O
O
N
O
O
H
H
N
N
CbzHN
H
O
4 (47%)
NH2 . TFA
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aReagents and conditions: (a) Et3N, DMF, 0 °C to rt, 16 h; (b) THF, EEDQ, rt, 16 h; (c) THF, 80
°C, 2 h; (d) TFAꢀCH2Cl2 (1:1, v/v), iceꢀbath, 15 min.
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Kinetic Evaluation of Probe Candidates. Benchmark kinetic values of KM, kcat and kcat/KM were
established for the twoꢀcomponent fluorogenic peptides using recombinant CTB, CTL and
cathepsin S (CTS). CTS, a lysosomal cysteine cathepsin also linked to cancer,13 was chosen to
evaluate probe specificity since the human enzyme is commercially available and has similar
substrate preferences as CTB and CTL. As shown in Table 1, CbzꢀPheꢀArgꢀAMC, CbzꢀArgꢀArgꢀ
AMC, 1 and 2 were excellent substrates of CTB as judged by their kcat/KM values. In contrast, CTL
and CTS efficiently hydrolyzed both CbzꢀPheꢀArgꢀAMC and probe 1 but had little activity towards
CbzꢀArgꢀArgꢀAMC and 2. These results indicate that CbzꢀLysꢀLys, similar to CbzꢀArgꢀArg, can
offer efficient recognition and maintain improved CTB specificity over CbzꢀPheꢀLys in selective
substrates.
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Prior to determining the kinetic parameters for the three component fluorogenic peptides, it was
necessary to demonstrate that 3 and 4 are chemically stable towards spontaneous hydrolysis and
AMC release under the assay conditions. As shown in the Figure 3, HPLC chromatograms
demonstrate that probes 3 and 4 are chemically stable in assay buffer for at least two hours,
indicating that the rates of spontaneous hydrolysis and AMC release are not significant and will not
impact the initial velocity measurements of the enzyme catalyzed reactions. In addition, the
chemical purity of the probes is high as only buffer components appear at early retention times in
each chromatogram.
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Figure 3: HPLC chromatograms demonstrating the chemical stability of the three component
peptides 3 and 4. Each probe (120 ꢁM) was dissolved in enzyme assay buffer and incubated at 37
ºC for the indicated length of time. An aliquot was taken and 90 ꢁL injected into an HPLC and
separated on a C18 reverse phase column. Over the course of two hours, the total peak area of
probes 3 and 4 remained constant and no AMC was observed at the expected retention times (2.3
minutes and 2.9 minutes respectively). Note that the early peaks in all chromatograms are from
buffer components and that the mobile phase used to separate 3 or 4 from AMC were different as
described in the materials and methods sections.
Once we were confident that probes 3 and 4 were chemically stable for enzyme kinetic analysis,
we determined how the PABA spacer affects recognition and turnover by CTB, CTL and CTS. The
introduction of PABA into probes 3 and 4 resulted in a lower apparent KM value for CTB when
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compared to the corresponding twoꢀcomponent peptides. This suggests that introduction of the
PABA linker results in higher affinity towards CTB and may improve probe performance in cellulo.
The lower KM value determined for 3 was partially offset by a lower kcat, but an impressive
specificity constant was maintained with kcat/KM = 61 mMꢀ1 sꢀ1 for CTB. Encouragingly, rapid
turnover of 4 was observed (kcat/KM = 231 mMꢀ1sꢀ1) resulting in the highest specificity constant of
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the novel CTB substrates tested. For comparison purposes, FꢀFHBG, with a kcat/kM = 43 mMꢀ1sꢀ1,
is a substrate that has been successfully used to image herpes simplex virus thymidine kinase
activity in humans. Surprisingly, CTL turnover of 3 suffered dramatically as no reliable kinetic data
was obtained even when the concentration of enzyme was increased 30 fold higher than used is
assays to evaluate compound 1. As expected, CTS efficiently hydrolyzed 3 while neither CTS nor
CTL efficiently processed probe 4.
Table 1: The kinetic parameters obtained for each fluorogenic peptide as a substrate of CTB,
CTL and CTS.a
Cathepsin B
Cathepsin L
Cathepsin S
kcat
(sꢀ1)
kcat/KM
(mMꢀ1sꢀ1)
kcat
(sꢀ1)
kcat/KM
(mMꢀ1sꢀ1)
kcat
kcat/KM
KM
(mM)
KM
(mM)
KM
(mM)
Probe
(sꢀ1) (mMꢀ1sꢀ1)
CbzꢀArgꢀ
ArgꢀAMC
0.024 ± 0.22 ±
0.34 ± 0.06
56 ± 3
165 ± 37
400 ± 70
9.2 ± 2.3
n.d.
n.d.
n.d.
0.005
0.01
0.52 ± 0.2
x 10ꢀ3
CbzꢀPheꢀ
ArgꢀAMC
0.38 ±
0.01
0.09 ± 0.01
0.19 ± 0.03
36 ± 2
26 ± 2
730 ± 280 0.072 ± 0.01 1.3 ± 0.1 18 ± 2
0.59 ± 0.12
x 10ꢀ3
0.39 ±
0.01
(1)
(2)
137 ± 13
148 ± 5
661 ± 149 0.088 ± 0.02 1.1 ± 0.1 13 ± 3
0.092 ± 0.14 ±
0.001 0.06
0.25 ± 0.01
37 ± 1
1.5 ± 0.6
n.d.
n.d.
n.d.
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(3)
(4)
0.082 ± 0.003 0.50 ± 0.04
61 ± 7
n.d.
n.d.
n.d.
0.056 ± 0.02 0.39 ± 0.03 69 ± 25
0.043 ± 0.11 ±
0.01 0.01
0.16 ± 0.04
37 ± 2
231 ± 70
2.5 ± 0.8
n.d.
n.d.
n.d.
aAll values were determined by an average of three trials ± standard deviation. Where indicated
by n.d. no reliable kinetic data could be obtained using concentration of enzyme as high as 10 nM.
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Cell Lysate Assays. Cell lysates were prepared from HeLa cervical cancer cells40 and incubated
individually with 100 ꢁM of CbzꢀArgꢀArgꢀAMC, 3, or 4 under acidic and reducing conditions
favoring lysosomal cysteine cathepsin activity. As shown in Figure 4, probe 4 was the most
efficient substrate consistent with the highest kcat/KM value. In contrast, the relative hydrolysis of
probe 3 was unexpectedly high with overall turnover similar to CbzꢀArgꢀArgꢀAMC. The higher
relative activity measured for probe 3 is likely due to the hydrolysis of this substrate by CTB
together with the additive contributions from other proteases present in the cell lysates.
To evaluate the cysteine cathepsin specificity of CbzꢀArgꢀArgꢀAMC, 3, and 4, cell lysates were
prepared from HeLa cells treated overnight with the wellꢀestablished CTB inhibitor CAꢀ074Me and
the broad spectrum cysteine cathepsin inhibitor E64d. Although CAꢀ074Me is routinely used to
efficiently inactivate CTB in living cells, it has been shown that CTL activity is also inhibited.41
Nonetheless, assays using cell lysates prepared from CAꢀ074Meꢀinhibited cells in combination with
the in vitro kinetic data using purified enzymes is a concise way to assess probe specificity. As
shown in Figure 4, CTB hydrolysis of CbzꢀArgꢀArgꢀAMC or 4 was not observed in lysates obtained
from cells treated with CAꢀ074Me or E64d. This demonstrates that both inhibitors effectively
inactivate CTB and that both CbzꢀArgꢀArgꢀAMC and 4 are highly specific to CTB over the other
cysteine cathepsins. In contrast, cell lysates prepared from cells treated with CAꢀ074Me maintained
about 35% of the relative activity towards probe 3 when compared to the lysates prepared from
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HeLa cells without inhibitor treatment. This clearly demonstrates that probe 3 is hydrolyzed by
CTB and one or more other enzymes. Lysates from HeLa cells treated with E64d were unable to
efficiently hydrolyze 3 demonstrating that the remaining 35% relative activity observed in lysates
prepared from the CAꢀ074Me treated cells results from other cysteine cathepsin activity.
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Figure 4: Cell Lysate assays to evaluate the specificity of 3 and 4 towards CTB. HeLa cell lysates
incubated with CbzꢀArgꢀArgꢀAMC, 3 and 4 using lysates prepared from untreated cells and cells
treated with CAꢀ074Me or E64d. The bars represent the relative activity (%) compared to CbzꢀArgꢀ
ArgꢀAMC incubated with untreated lysates adjusted to total protein concentration. For each
experiment, background RFU due to spontaneous hydrolysis in wells containing no enzyme was
subtracted from the total fluorescence and error bars depict standard deviation from 4 independent
trials.
Cancer Cells Assays. Next, the effectiveness of the twoꢀcomponent fluorogenic peptides to
detect intracellular protease activity over short incubation times was evaluated. In separate wells,
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live HeLa cells were incubated with CbzꢀArgꢀArgꢀAMC, 1, or 2. Following incubation, the cells
were washed twice with phosphate buffered saline (PBS) to remove unreacted probe in the media or
probe absorbed onto the surface of the cancer cells. The cells were then placed in a fluorescence
microscope and images taken. As shown in Figure 5, none of the compounds tested resulted in the
detectable accumulation of fluorescence inside of living cells over the two hour incubation time.
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Figure 5: Fluorescence Microscope Images of HeLa cells incubated with the twoꢀcomponent
peptides. Fluorescent and bright field microscope images taken of HeLa cells treated with 40 ꢁM of
a) CbzꢀArgꢀArgꢀAMC b) twoꢀcomponent peptide (1) c) twoꢀcomponent peptide (2). Images were
recorded using a fluorescence microscope after treatment of cells for two hours with the appropriate
compound followed by two washes with PBS buffer to remove unreacted substrate in the media and
absorbed onto the cell surface.
To evaluate the utility of the three component probe candidates for visualizing intracellular
protease activity, each peptide was incubated at 40 ꢁM in the presence of living HeLa cells
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followed by washing twice with PBS buffer to remove unreacted probe and probe absorbed onto the
cell surface. As shown in Figure 6 a) and b), strong fluorescence intensity was clearly visualized
inside of HeLa cells. Interestingly, cells incubated with probe 3 underwent rapid morphological
changes reminiscent of cell death while those treated with 4 appeared to tolerate this compound
well. To evaluate the performance of each three component probe in another cancer type, a Her2ꢀ
positive breast cancer cell line derived from MDꢀMBAꢀ231 called H2N42 was incubated with 3 or 4.
Intracellular fluorescence was observed in this cell line consistent with images taken of the HeLa
cells (see Figures 6c and 6d).
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Figure 6: Bright Field and Fluorescence Microscope Images of HeLa cervical and MBꢀMDAꢀ231ꢀ
H2N breast cancer cells lines treated with probes 3 and 4. Fluorescent microscope images taken of
HeLa cervical cancer cells treated for 2 hours with a) 40 ꢁM of (3) and b) 40 ꢁM of (4) and MBꢀ
MDAꢀ231ꢀH2N breast cancer cells treated c) 40 ꢁM of (3) and d) 40 ꢁM of (4). Control
experiments to evaluate probe specificity were conducted by overnight treatment with CAꢀ074Me
(10 ꢁM CTB inhibitor) or E64d (10 ꢁM broad spectrum cysteine cathepsin inhibitor) followed by
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incubation with each probe for 2 hours, washed twice with PBS then imaged. White bars represent
50 ꢁm.
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To evaluate the specificity of probe candidates towards CTB in live cells, CAꢀ074Me was used to
treat both cervical and breast cancer cell lines. As shown in Figure 6, HeLa and H2N cells treated
with CAꢀ074Me or E64d were unable to efficiently activate probe 4, suggesting high CTB
specificity of this probe in live cancer cells. In contrast, cells treated first with CAꢀ074Me followed
by probe 3 showed residual intracellular fluorescence, indicating that 3 is hydrolyzed inside the
CTBꢀinhibited cells consistent with the kinetic and lysate data that 3 is an efficient substrate of one
or more other enzymes. Indeed, the broad spectrum cysteine cathepsin inhibitor E64d prevented the
onset of fluorescence in HeLa and H2N cells, supporting the hypothesis that turnover of 3 results
from another cysteine protease such as CTS.43 Nonetheless, these experiments demonstrate that
designing future CTB imaging probes based on CbzꢀPheꢀLysꢀPABA may lack the desired
specificity for selectively sensing CTB activity in living cells.
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Cell Viability and Proliferation Assays.
To evaluate changes in cell viability of the HeLa and H2N cell lines induced by probes 3 and 4,
we used the 3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5 diphenyl tetrazolium bromide (MTT) assay. MTT is a
yellow tetrazolium salt that is reduced to an insoluble purple formazan dye by reductase enzymes in
viable cells. Therefore, cell death through necrosis and/or apoptosis induced by the probe
candidates can be quantified using this convenient spectrophotometric assay. As shown in Figure 7,
a significant decrease in cell viability was found in HeLa and H2N cells treated with probe 3 at 10
and 40 ꢁM. Interestingly, the H2N cells had no decrease in viability at 1 ꢁM of probe 3 while HeLa
cell viability was reduced to approximately 50% compared to the untreated cells. This MTT assay
confirms the high toxicity of probe 3 in these cells lines, as observed in the fluorescence microscopy
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experiments, preventing its use for live cell applications. In contrast, HeLa and H2N cells treated
with CbzꢀArgꢀArgꢀAMC or probe 4, no statistically significant differences in cell viability were
observed at 1, 10 ꢁM and 40 ꢁM concentrations. This data confirms that the fluorescence
microscopy images in Figure 6 were performed on a viable population of HeLa and H2N cells with
no significant increases in necrosis or apoptosis induced by 4 throughout the two hour incubation
times.
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Figure 7: The effects of CbzꢀArgꢀArgꢀAMC, 3 and 4 on cell viability. HeLa and H2N cells were
treated with probes 3, 4 or CbzꢀArgꢀArgꢀAMC at 1, 10 and 40 ꢁM. Cell viability was quantified
using MTT and expressed as a % of the viability of untreated cells. Statistical differences in
viability were determined by performing ANOVA Posthoc dunnett’s test; p<0.05 was considered
significant and indicated by ***.
CONCLUSION
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The purpose of this study was to determine if PABA derivatized peptides were potentially useful
in the development of molecular imaging probes specific to Cathepsin B. Kinetic studies using
purified enzyme revealed that the incorporation of PABA into the fluorogenic peptides produced
highly efficient threeꢀcomponent substrates of CTB. Surprisingly, CTL did not tolerate the
presence of the PABA linker in probe 3, a compound expected to be an excellent substrate of this
enzyme. Although compound 3 is an efficient substrate of CTB, low specificity and high toxicity
prevented its use for live cell fluorescence microscopy studies. Probe 4 is an easily synthesized,
highly efficient substrate capable of selectively assessing CTB activity in cell lysates under
conditions favoring cysteine cathepsin activity. Fluorescence microscopy cell assays indicate that 4
is cell permeable and can efficiently reveal protease activity in viable cervical and breast cancer cell
lines. Consistent with the cell lysate assays, the onset of fluorescence in cells treated with 4 can be
blocked using the known CTB inactivator CAꢀ074Me. To our knowledge, 4 is the first reported
example of a small molecule fluorogenic substrate selective towards CTB over other lysosomal
cysteine cathepsins having utility for detecting CTB activity inside live cells. Current efforts in our
laboratory seek to synthesize probes using CbzꢀLysꢀLysꢀPABA as a CTB recognition peptide
conjugated to a radioactive reporter or fluorophores excited at longer wavelengths for detailed
cancer cell assays and in vivo molecular imaging.
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EXPERIMENTAL SECTION
General Procedures. Proton (1H) and carbon (13C) NMR spectra were acquired at the Lakehead
University Instrumentation Laboratory (LUIL) on a Varian Unity Inova 500 MHz spectrometer in
DMSOꢀd6 with TMS as the internal standard, where J (coupling constant) values are estimated in
hertz (Hz). Microanalyses were measured for C, H, N using Elementar Vario EL and were within ±
0.4% of theoretical values indicating that all compounds were >95% pure. Thin layer
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chromatography (TLC) and silica gel column chromatography were performed using TLC Silica
Gel 60 F254 (EMD) and SiliaFlash®P60 (SiliCycle), respectively. All other reagents and solvents
6
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9
were used as purchased without further purification. Human Cathepsin B was purchased from Sino
Biological Inc. (China), human Cathepsin L from Calbiochem and human Cathepsin S from
Novoprotein with kinetic studies performed in a Biotek Synergy 4 plate reader. HPLC was
performed on a Agilent 1200 Infinity LC with compounds separated on a Agilent Eclipse XDBꢀC18
column (4.6 mm, 150 mm, 5 µm) and detected using 1200 series variable wavelength detector
(G1314B). All cell culture reagents were purchased from Fisher Scientific. The stock solutions of
all peptides were made with DMSO but did not exceed 1% v/v in the media used for the cells
assays. Fluorescence was visualized and digitally captured using an inverted fluorescent
microscope (Zeiss Axiovert 200 or the Olympus TL4, filter set with excitation maxima 365 nm and
emission wavelength > 420 nm) and digital camera attachment (QImaging QICAM Q21310).
Compounds 5,36 6/8,44 10,45 1146 and 1547 were prepared according to literature procedures.
CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀAMC (7): To a solution of 5 (212 mg, 0.52 mmol) in THF – H20
(3.5:1.5 v/v, 5 mL) and NaHCO3 (44 mg, 0.52 mmol) was added 6 (206 mg, 0.52 mmol) in THF
(3.5 mL) and stirred at room temperature for 16 h. The reaction mixture was then concentrated in
vacuo and extracted with ethyl acetate (15 mL x 3). The combined organic phase was washed with
brine (25 mL) and dried (Na2SO4). After filtration, the solvent was removed from filtrate and the
crude residue was purified by silica gel column chromatography using dichloromethane – methanol
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1
(9:1 v/v) as eluent to obtain the pure title compound 7. White solid, 288 mg (81%); H NMR
(DMSOꢀd6) δ 1.28ꢀ1.45 (m, 4H), 1.35 (s, 9H), 1.60ꢀ1.70 (m, 1H), 1.70ꢀ1.81 (m, 1H), 2.41 (s, 3H),
2.75 (dd, J = 10.9, 13.7 Hz, 1H), 2.83ꢀ2.95 (m, 2H), 3.05 (dd, J = 3.6, 15.3 Hz, 1H), 4.31ꢀ4.39 (m,
1H), 4.39ꢀ4.46 (m, 1H), 4.96 (s, 2H), 6.28 (d, J = 1.3 Hz, 1H), 6.79 (t, J = 5.6 Hz, 1H), 7.16ꢀ7.36
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(m, 10H), 7.47ꢀ7.54 (m, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 1.9 Hz, 1H), 8.33 (d, J = 7.6 Hz,
1H), 10.52 (s, 1H). 13C NMR (DMSOꢀd6) δ 18.5, 23.3, 28.7, 29.8, 32.2, 37.9, 54.2, 56.5, 65.7, 77.8,
106.2, 112.8, 115.6, 115.7, 126.4, 126.7, 127.3, 127.9, 128.2, 128.5, 128.8, 129.7, 137.5, 138.5,
142.7, 153.5, 154.1, 156.0, 156.4, 160.5, 171.9, 172.3. Elemental analysis calculated (%) for
C38H44N4O8: C, 66.65; H, 6.48; N, 8.18. Found: C, 66.40; H, 6.74; N, 8.10.
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CbzꢀPheꢀLysꢀAMC . TFA (1): To a solution of trifluoroacetic acid – DCM (1:1 v/v, 2 mL) at
iceꢀbath temperature was added 7 (200 mg, 0.29 mmol) and stirred for 15 min. After reaction
diethyl ether was added to the reaction mixture to precipitate out the solids. Then the solids were
centrifuged out and successively washed with diethyl ether (5 mL x 2) and ethyl acetate (5 mL x 2),
and dried under high vacuum in the dark to get the pure title compound 1. White solid, 204 mg
1
(quant); H NMR (DMSOꢀd6) δ 1.23ꢀ1.80 (m, 6H), 2.33 (s, 3H), 2.61ꢀ2.78 (m, 3H), 2.94ꢀ3.04 (m,
1H), 4.23ꢀ4.32 (m, 1H), 4.32ꢀ4.43 (m, 1H), 4.89 (s, 2H), 6.21 (d, J = 1.2 Hz, 1H), 7.08ꢀ7.31 (m,
10H), 7.45 (dd, J = 2.1, 8.6 Hz, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.70ꢀ7.86
(m, 4H), 8.38 (d, J = 7.6 Hz, 1H), 10.54 (s, 1H). 13C NMR (DMSOꢀd6) δ 18.5, 22.9, 27.2, 31.8,
37.8, 45.0, 54.1, 56.5, 65.7, 106.2, 112.8, 115.6, 115.8, 117.7 (J = 300 Hz), 126.4, 126.7, 127.9,
128.2, 128.5, 128.8, 129.7, 137.5, 138.5, 142.6, 153.6, 154.1, 156.4, 158.9 (J = 31 Hz), 160.5,
171.8, 172.3. Elemental analysis calculated (%) for C35H37F3N4O8: C, 60.17; H, 5.34; N, 8.02.
Found: C, 59.82; H, 5.52; N, 7.95.
CbzꢀLysꢀNꢀεꢀBOCꢀLysꢀNꢀεꢀBOCꢀAMC (9): To a solution of 8 (592 mg, 1.24 mmol) in dry
DMF (5 mL) 5 (500 mg, 1.24 mmol) and triethylamine (173 µL, 1.24 mmol) were added at 0 °C
followed by stirring for 30 min. The solution was allowed to slowly reach room temperature and
the reaction stirred for another 16 h. The reaction mixture was then evaporated in vacuo and the
residue was dissolved in ethyl acetate (20 mL) and successively washed with 1 M citric acid (10 mL
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x 2), brine (15 mL) and dried (Na2SO4). After filtration, the solvent was removed from filtrate and
the crude residue was purified by silica gel column chromatography using ethyl acetate – hexanes
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(4:1 v/v) as an eluent to obtain the pure title compound 9. White solid, 935 mg (98%); H NMR
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(DMSOꢀd6) δ 1.29ꢀ1.44 (m, 6H), 1.37 (s, 18H), 1.45ꢀ1.79 (m, 6H), 2.40 (s, 3H), 2.79ꢀ2.95 (m, 4H),
3.96ꢀ4.08 (m, 1H), 4.33ꢀ4.42 (m, 1H), 5.04 (s, 2H), 5.53ꢀ 5.61 (m, 1H), 6.28 (s, 1H), 6.77 (t, J = 5.0
Hz, 2H), 7.26ꢀ7.45 (m, 5H), 7.49 (dd, J = 2.0, 8.5 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 2.0
Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H), 10.45 (s, 1H). 13C NMR (DMSOꢀd6) δ 18.4, 23.2, 23.3, 24.9,
25.8, 28.7, 28.7, 29.7, 29.7, 32.1, 33.8, 54.0, 55.1, 65.9, 77.8, 77.8, 106.1, 112.8, 115.5, 115.7,
126.4, 128.2, 128.2, 128.8, 137.5, 142.6, 153.5, 154.1, 156.0, 156.5, 157.1, 160.5, 172.0, 172.7.
Elemental analysis calculated (%) for C40H55N5O10: C, 62.73; H, 7.24; N, 9.14. Found: C, 63.12; H,
7.10; N, 8.92.
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CbzꢀLysꢀLysꢀAMC.2TFA (2): To a solution of trifluoroacetic acid – DCM (1:1 v/v, 1 mL) at
iceꢀbath temperature was added 9 (100 mg, 0.13 mmol) and stirred for 15 min. After reaction,
diethyl ether was added to the mixture to precipitate out the solids. Then the suspension was
centrifuged and successively washed with diethyl ether (5 mL x 2) and ethyl acetate (5 mL x 2), and
dried under high vacuum in the dark to get the pure title compound 2. White solid, 60 mg (58%). 1H
NMR (DMSOꢀd6) δ 1.28ꢀ1.83 (m, 12H), 2.41 (s, 3H), 2.69ꢀ2.84 (m, 4H), 3.98ꢀ4.11 (m, 1H), 4.34ꢀ
4.47 (m, 1H), 5.04 (s, 2H), 6.28 (d, J = 1.2 Hz, 1H), 7.27ꢀ7.56 (m, 7H), 7.67ꢀ7.94 (m, 8H), 8.23ꢀ
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8.34 (m, 1H), 10.59 (s, 1H). C NMR (DMSOꢀd6) δ 18.5, 22.9, 22.9, 27.1, 27.2, 31.7, 31.7, 39.1,
45.0, 53.9, 54.8, 65.9, 106.2, 114.1, 115.6, 115.7, 117.7 (J = 300 Hz), 126.5, 128.2, 128.3, 128.8,
137.5, 142.6, 153.6, 154.1, 156.5, 158.7 (J = 31 Hz), 160.5, 171.9, 172.7. Elemental analysis
calculated (%) for C34H41F6N5O10.H2O: C, 50.31; H, 5.34; N, 8.63. Found: C, 50.58; H, 5.35; N,
8.79
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CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀPABꢀOH (12): A solution of 11 (0.83 g, 1.57 mmol), PABA (232 mg,
1.89 mmol) in THF (15 mL) was prepared followed addition of EEDQ (467 mg, 1.89 mmol). After
stirring for 16 hours at room temperature, the solvent was removed under reduced pressure and the
resulting residue taken into diethyl ether (25 mL) to form a suspension. The solid was filtered out
and washed with diethyl ether (25 mL x 2) and dried under vacuum to get the pure title compound
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12 as a pale yellow solid, 0.93 g (94%). H NMR (DMSOꢀd6) δ 1.20ꢀ1.50 (m, 4H), 1.36 (s, 9H),
1.50ꢀ1.60 (m, 1H), 1.60ꢀ1.80 (m, 1H), 2.74 (dd, J = 11, 13.5 Hz, 1H), 2.80ꢀ2.95 (m, 2H), 3.03 (dd, J
= 5.5, 13.5 Hz, 1H), 4.30ꢀ4.37 (m, 1H), 4.37ꢀ4.50 (m, 1H), 4.44 (d, J = 18 Hz, 2H), 4.96 (s, 2H),
5.12 (t, J = 5.5 Hz, 1H), 6.79 (t, J = 5.5 Hz, 1H), 7.15ꢀ7.35 (m, 12H), 7.51 (d, J = 8.5 Hz, 1H), 7.56
(d, J = 8.0 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 10.01 (s, 1H). 13C NMR (DMSOꢀd6) δ 23.2, 28.7, 29.7,
32.5, 37.9, 40.5, 53.9, 56.5, 63.1, 65.7, 77.8, 119.5, 126.7, 127.4, 127.9, 128.1, 128.5, 128.8, 128.7,
137.5, 137.9, 138.0, 138.5, 156.0, 156.3, 170.8, 172.0. Elemental analysis calculated (%) for
C35H44N4O7: C, 66.44; H, 7.01; N, 8.85. Found: C, 66.32; H, 7.23; N, 8.84.
CbzꢀPheꢀLysꢀNꢀεꢀBOCꢀPABCꢀAMC (14): AMC (0.25 g, 1.43 mmol) was taken in a 25 mL
flask and a solution of 15% phosgene in toluene 10 mL was added. The reaction mixture was
refluxed at 120 °C for 16 h under argon. After reaction, the mixture was cooled to room temperature
and argon was bubbled into the solution for 10 minutes to remove unreacted phosgene gas. Then the
solvents were removed under reduced pressure to dryness yielding 7ꢀisocyanatoꢀ4ꢀmethylchromenꢀ
2ꢀone (13) as a white powdered solid (ca. 0.29 g) which was used immediately without further
purification. A solution of isocyanate 13 (ca. 0.29 g, 1.43 mmol) and 12 (0.6 g, 0.95 mmol) in dry
THF (5 mL) was stirred at 80 °C under argon for 2 h. After reaction, solvent was removed under
reduced pressure to give the crude product 14 which contained unreacted isocyanate 13 and alcohol
12. To remove the isocyanate 13, the crude product was dissolved in dichloromethane (25 mL) and
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