Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of the Cysteine Protease Cathepsin L

Article abstract of DOI:10.1021/acs.inorgchem.8b00978

Two new Re(I)- and Ru(II)-based inhibitors were synthesized with the formulas [Re(phen)(CO)₃(1)](OTf) (7; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru(bpy)₂(2)](Cl)₂ (8; bpy = 2,2′-bipyridine), where 1 and 2 are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds 7 and 8 were characterized using various spectroscopic techniques and elemental analysis; 7 and 8 both show exceptionally long excited state lifetimes. Re(I)-based complex 7 inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor 1 with a second-order rate constant of 211000 ± 42000 M^-1 s^-1. Irreversible ligation of 7 to papain, a model of CTSL, was analyzed with mass spectroscopy, and the major peak shown at 24283 au corresponds to that of papain-1-Re(CO)₃(phen). Compound 7 was well tolerated by DU-145 prostate cancer cells, with toxicity evident only at high concentrations. Treatment of DU-145 cells with 7 followed by imaging via confocal microscopy showed substantial intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of 7 to label CTSL in living cells. Overall this study reveals that a Re(I) complex can be attached to an enzyme inhibitor and enhance potency and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due to long excited state lifetimes and non-native element composition.

Full text of DOI:10.1021/acs.inorgchem.8b00978

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Affinity-Enhanced Luminescent Re(I)- and Ru(II)-Based Inhibitors of
the Cysteine Protease Cathepsin L
Matthew Huisman,† Jacob P. Kodanko,† Karan Arora,† Mackenzie Herroon,‡ Marim Alnaed,†
John Endicott,† Izabela Podgorski,‡,§ and Jeremy J. Kodanko*,†,§
†Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
‡Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201, United States
§Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, United States
S
* Supporting Information
ABSTRACT: Two new Re(I)- and Ru(II)-based inhibitors were synthesized
with the formulas [Re(phen)(CO)3(1)](OTf) (7; phen = 1,10-phenanthroline,
OTf = trifluoromethanesulfonate) and [Ru(bpy)2(2)](Cl)2 (8; bpy = 2,2′-
bipyridine), where 1 and 2 are the analogues of CLIK-148, an epoxysuccinyl-
based cysteine cathepsin L inhibitor (CTSL). Compounds 7 and 8 were
characterized using various spectroscopic techniques and elemental analysis; 7
and 8 both show exceptionally long excited state lifetimes. Re(I)-based complex
7 inhibits CTSL in the low nanomolar range, affording a greater than 16-fold
enhancement of potency relative to the free inhibitor 1 with a second-order rate
constant of 211000
42000 M−1 s−1. Irreversible ligation of 7 to papain, a
model of CTSL, was analyzed with mass spectroscopy, and the major peak
shown at 24283 au corresponds to that of papain-1-Re(CO)3(phen). Compound
7 was well tolerated by DU-145 prostate cancer cells, with toxicity evident only
at high concentrations. Treatment of DU-145 cells with 7 followed by imaging via confocal microscopy showed substantial
intracellular fluorescence that can be blocked by the known CTSL inhibitor CLIK-148, consistent with the ability of 7 to label
CTSL in living cells. Overall this study reveals that a Re(I) complex can be attached to an enzyme inhibitor and enhance potency
and selectivity for a medicinally important target, while at the same time allowing new avenues for tracking and quantification due
to long excited state lifetimes and non-native element composition.
such as fluorophores for detecting active cysteine cathepsins,
including CTSL, have applications in in vivo and ex vivo
detection and diagnosis of human diseases.15−17
INTRODUCTION
■
Cysteine cathepsins are proteases that play a major role in
normal cellular physiology and also in pathogenesis. A total of
11 family members of cysteine cathepsins have been
characterized to date.1 Aberrant activity and overexpression of
cysteine cathepsins are associated with many human disease
states.2,3 Because of the crucial role of these proteases in
biology, their inhibitors have been pursued aggressively by
academic laboratories and also by pharmaceutical companies, as
chemical tools to understand the role of cysteine proteases in
biology and also as pharmaceuticals.4−6
Cathepsin L (CTSL) is a lysosomal cysteine protease that is
upregulated in some cancers,7 neurodegenerative disorders,
atherosclerosis,8,9 and inflammation.10,11 Because of its higher
expression levels in diseased tissues, CTSL can be used for
diagnostic purposes. For instance, cysteine proteases are
significantly more abundant in malignant vs benign glioma
tumors where, in contrast to cathepsin B (CTSB) found in the
tumor and tumor-associated endothelial cells, immunohisto-
chemical staining reveals selective localization of CTSL to
tumor cells only.12 Overall, CTSL inhibitors have significant
potential therapeutic value, especially as adjuvants to current
therapies.7,13,14 In addition, small molecules that carry reporters
Research in the area of cysteine protease inhibition has been
dominated by purely organic compounds.4,5,14,18,19 Most
inhibitors were designed to carry a reactive functional group
or “warhead” that creates a covalent bond with the enzyme
upon attack by the active site cysteine thiolate. This includes
epoxysuccinyl-based inhibitors of CTSL, such as CLIK-148
(Figure 1), which block the action of this protease in vitro and
in vivo through selective binding and covalent modification by
epoxide-opening reactions.4,20 Metal-based protease inhibitors
are much more rare,21 although good progress has been made
identifying Pd(II),22,23 Au(III),22,24 and Re(V)25,26 complexes
with low micromolar to nanomolar potentcies against cysteine
proteases such as CTSB. In particular, Au(III)-based
compounds that interact directly with the active site thiolate
of cysteine proteases have shown activity in preclinical cancer
models.27,28 For all of these compounds, the metal is
Received: April 12, 2018
© XXXX American Chemical Society
A
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stirred overnight for 18 h. After consumption of the starting material,
as judged by TLC analysis, the reaction mixture was filtered through
Celite. The solvent was evaporated in vacuo to obtain the p-
nitrophenol ester as a crude yellow solid (675 mg). The p-nitrophenol
ester was analyzed by 1H NMR and TLC, showing only p-nitrophenol
as a minor impurity, and was used without further purification. Data
1
for the ester are as follows: Rf = 0.4, silica, 1/1 hexane/EtOAc; H
NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.9 Hz, 2H), 7.35−7.29 (m,
5H), 7.17 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 8.9
Hz, 2H), 5.16 (dd, J = 14.6, 8.1 Hz, 1H), 3.78 (d, J = 2.0 Hz, 1H), 3.50
(d, J = 2.0 Hz, 1H), 3.08−3.02 (m, 1H), 2.99−2.96 (m, 1H), 2.93 (s,
3H), 2.76 (s, 3H).
The crude p-nitrophenol ester (358 mg, 0.837 mmol) in dry
CH2Cl2 (6 mL) was maintained at room temperature for 5 min under
a nitrogen atmosphere. N-(3-Aminopropyl)-[2,2′-bipyridine]-5-car-
boxamide (5; 195 mg, 0.761 mmol) in CH2Cl2 (6 mL) was added
dropwise over a period of 20 min. The reaction mixture was
maintained under a nitrogen atmosphere for 16 h at room
temperature. After consumption of the starting material, as judged
by TLC analysis, the organic layer was evaporated in vacuo to give a
crude mixture which was purified by silica gel chromatography (0−
20% MeOH/EtOAc) to afford the product 2 as a white solid (244 mg,
Figure 1. Structures of epoxysuccinyl-based cysteine cathepsin
inhibitors.
considered to be the warhead that binds directly to the active
site thiolate.
Re(I) and Ru(II) fluorogenic metal fragments have many
applications in the labeling and detection of biomolecules.29,30
These fluorophores are attractive because they show several
advantages over more traditional organic emitters, including
long-lived excited states that can be used in time-gated imaging
experiments, enhanced resistance to photobleaching, and
compositions containing non-native elements, making detec-
tion and quantification by ICP-MS straightforward.29,31,32 In
addition, Re(I) tricarbonyl compounds can be tracked by IR-
based spectromicroscopy, a new type of cell imaging.33 While
tagging biologically active molecules with fluorophores allows
for visualization and tracking targets of interest, the fluorophore
has traditionally provided no real advantage toward gaining
higher potency or selectivity between related targets.
1
59% over two steps). Data for 2 are as follows: H NMR (400 MHz,
CD3OD) δ 9.08 (d, J = 1.5 Hz, 1H), 8.68 (d, J = 4.4 Hz, 1H), 8.41 (m,
2H), 8.32 (dd, J = 8.3, 2.5 Hz, 1H), 7.96 (dt, J = 7.8, 1.5 Hz, 1H), 7.47
(dd, J = 4.9, 2.5 Hz, 1H), 7.31−7.28 (m, 2H), 7.25−7.21 (m, 3H),
5.08 (t, J = 7.3 Hz, 1H), 3.61 (d, J = 2.0 Hz, 1H), 3.47−3.43 (m, 3H),
3.35−3.32 (m, 2H), 3.06−3.01 (m, 1H), 2.98−2.92 (m, 1H), 2.84 (s,
3H), 2.82 (s, 3H), 1.82 (p, J = 6.9 Hz, 2H); [α]D25 +51 (c = 1.0,
MeOH); 13C NMR (100 MHz, CD3OD) δ 171.2, 167.4, 166.6, 166.5,
158.0, 154.9, 149.1, 148.0, 137.3, 136.2, 135.9, 129.9, 129.0, 128.1,
126.7, 124.4, 121.6, 120.4, 53.4, 53.1, 50.6, 37.7, 36.9, 36.1, 34.7, 28.6;
IR (thin film) 3278, 3063, 2934, 1629, 1589, 1538, 1456, 1435, 1297,
1249, 1145, 1091, 1023, 992, 893, 807, 759, 700, 639, 618, 523, 496
cm−1; HRMS (ESMS) calculated for C29H32N6O5 [M + H]+ 545.2434,
found 545.2512.
In this paper, we report CTSL inhibitors bearing Re(I) and
Ru(II) metal centers that not only luminesce but also show
enhanced affinity and selectivity for inhibition of CTSL over
other enzymes in this family. Importantly, this study reveals a
new strategy for using coordinatively saturated metal complexes
to enhance potency through favorable noncovalent interactions
with protein targets. Both compounds show significantly longer
lived excited states in comparison to more traditional organic
fluorophores, which makes them appropriate for time-gated
imaging experiments. Furthermore, we report a Re(I)-based
inhibitor that is cell permeable and nontoxic at nanomolar and
low micromolar concentrations. Our data are consistent with
this Re(I) inhibitor labeling CTSL in living prostate cancer
cells.
Methyl [2,2′-Bipyridine]-5-carboxylate (4). A solution of methyl 6-
chloronicotinate (1.00 g, 5.83 mmol) and pyridin-2-ylzinc(II) bromide
(17.48 mL, 8.74 mmol, 0.5 M in THF) was maintained at room
temperature, and argon was bubbled through the solution for 20 min.
Tetrakis(triphenylphosphine)palladium(0) (337 mg, 0.291 mmol) was
added to the mixture, and the vessel with this mixture was sealed under
an inert atmosphere, wrapped in aluminum foil, and heated to 65 °C
for 16 h. The reaction mixture was a tan-yellow suspension which was
poured into aqueous 10% EDTA solution (50 mL) and stirred for 15
min, and then saturated Na2CO3 was added to achieve pH ∼8. The
product was extracted with CH2Cl2 (3 × 28 mL) and dried over
Na2SO4, and solvent was evaporated in vacuo to obtain the crude
product. The crude mixture was purified by silica gel chromatography
(0−10% EtOAc/hexanes) to afford 4 as a white solid (1.01 g, 81%).
1H NMR and ESMS spectral data for 4 were in good agreement with
data from the literature.35
EXPERIMENTAL SECTION
N-(3-Aminopropyl)-[2,2′-bipyridine]-5-carboxamide (5). A solu-
tion of 4 (1.01 g, 4.71 mmol) and propane-1,3-diamine (4.79 mL, 4.25
g 55.2 mmol) in MeOH (80 mL) was refluxed for 18 h under a
nitrogen atmosphere. After consumption of the starting material, as
judged by TLC analysis, the reaction mixture was concentrated under
reduced pressure and excess 1,3-propanediamine was removed by
azeotropic distillation from toluene (3 × 60 mL) to give compound 5
as a white solid (1.01 g 83%). Data for 5 are as follows: 1H NMR (400
MHz, CD3OD) δ 9.07 (d, J = 1.5 Hz, 1H), 8.68 (d, J = 4.4 Hz, 1H),
8.43−8.40 (m, 2H), 8.31 (dd, J = 8.3, 2.5 Hz, 1H), 7.96 (dt, J = 7.8,
1.5 Hz, 1H), 7.47 (dd, J = 4.9, 2.5 Hz, 1H), 3.50 (t, J = 6.9 Hz, 2H),
2.77 (t, J = 6.9 Hz, 2H), 1.82 (p, J = 6.9 Hz, 2H); 13C NMR (100
MHz, CD3OD) δ 166.6, 158.0, 154.9, 149.1, 148.0, 137.3, 135.9, 129.9,
124.4, 121.6, 120.4, 38.2, 36.9, 31.4; IR (thin film): 3294, 3060, 3003,
2952, 2919, 2872, 1629, 1589, 1536, 1479, 1456, 1434, 1422, 1367,
1331, 1287, 1249, 1151, 1121, 1089, 1015, 995, 860, 849, 756, 664,
637, 617, 592, 510, 492 cm−1; HRMS (ESMS) calculated for
C14H16N4O [M + H]+ 257.1324, found 257.1400.
■
General Considerations. NMR spectra were recorded on a
Varian FT-NMR Mercury 400 MHz spectrometer. Mass spectra were
recorded on a time-of-flight Micromass LCT Premier XE spectrom-
eter. IR spectra were recorded on a Nicolet FT-IR spectrophotometer
(thin film). UV−vis spectra were recorded on a Varian Cary 50
spectrophotometer. All reactions were performed an under ambient
atmosphere unless otherwise noted. Anaerobic reactions were
performed by purging the reaction solutions with Ar or N2.
Synthesis. (2S,3S)-N2-(3-([2,2′-Bipyridine]-5-carboxamido)-
propyl)-N3-(1-(dimethylamino)-1-oxo-3-phenylpropan-2-yl)-
oxirane-2,3-dicarboxamide (2). A solution of (2S,3S)-3-(((S)-1-
(dimethylamino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)oxirane-2-car-
boxylic acid (6;34 477 mg, 1.56 mmol) and p-nitrophenol (217 mg,
1.56 mmol) in EtOAc (4.5 mL) was maintained at 0 °C for 5 min
under a nitrogen atmosphere. A solution of DCC (331 mg, 1.60
mmol) in EtOAc (4.5 mL) was added dropwise over a period of 30
min. The reaction mixture was warmed to room temperature and
B
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[Re(Phen)(CO)3(1)](OTf) (7). In a sealable tube, a solution of 134
(191 mg, 0.470 mmol) and CHCl3/MeOH (4.5/18 mL) was
maintained at room temperature and argon was bubbled through
the solution for 20 min. Re(1,10-phenanthroline)(CO)3(OTf)36 (210
mg, 0.350 mmol) was added to the mixture, and the tube containing
the mixture was sealed under an inert atmosphere, wrapped in
aluminum foil, and heated to 60 °C for 16 h. The solvent was removed
in vacuo without external heating, resulting in a yellow solid. The
resulting 419 mg of solid was purified using silica gel chromatography
(0−10% MeOH/CH2Cl2) to give a yellow solid after solvent removal:
178 mg; Rf = 0.4, silica gel, 9/1 CH2Cl2/MeOH. The precipitate was
dissolved in a small amount of MeOH (0.5 mL) and layered with Et2O
(10 mL). After this solution was stored at −20 °C for 16 h, the
complex precipitated as a yellow solid. The solution was decanted, and
layering was repeated two more times. The solid was isolated by
scraping and dried in vacuo to provide Re(Phen)(CO)3(1)(OTf) (7)
as a microcrystalline yellow solid (162 mg, 50%). Data for 7 are as
follows: mp 137 °C; 1H NMR (400 MHz, CD3OD) δ 9.73 (d, J = 5.4
Hz, 2H), 8.93−8.90 (m, 2H), 8.29 (d, J = 6.4 Hz, 2H), 8.20−8.16 (m,
4H), 7.32−7.21 (m, 5H), 7.11 (d, J = 6.4 Hz, 2H), 5.06 (t, J = 7.3 Hz,
1H), 3.28−3.26 (m, 2H), 3.25 (d, J = 1.5 Hz, 1H), 3.14 (d, J = 1.5 Hz,
1H), 3.04 (dd, J1 = 13.4 Hz, J2 = 7.2 Hz, 1H), 2.94 (dd, J1 = 13.4 Hz, J2
= 7.7 Hz, 1H), 2.88 (s, 3H), 2.85 (s, 3H), 2.68 (t, J = 6.6 Hz, 2H); IR
(thin film) 3547, 3296, 3065, 2934, 2029, 1906, 1678, 1639, 1520,
1429, 1281, 1223, 1152, 1028, 897, 849, 723, 702, 637, 573, 540, 517,
484, 420 cm−1; HRMS (ESMS) calcd for C38H34N6O7Re [M]+
861.2046, found 861.1794; UV−vis λmax 275 nm (ε = 33300 M−1
cm−1), λem 543 nm. Anal. Calcd for C38H34F3N6O10ReS (7·2H2O): C,
43.63; H, 3.66; N, 8.03. Found: C, 43.89; H, 3.62; N, 7.92.
[Ru(bpy)2(2)](Cl)2 (8). In a sealable tube, a solution of 2 (40 mg,
0.074 mmol) and EtOH (15 mL) was maintained at room temperature
and argon was bubbled through the solution for 20 min. The
compound cis-[Ru(bpy)2Cl2]37 (30 mg, 0.062 mmol) was added, and
the tube containing the mixture was sealed under an inert atmosphere,
wrapped in aluminum foil, and heated to 80 °C for 16 h, during which
time the mixture turned from dark violet to bright orange. After the
mixture was cooled to room temperature, the solvent was removed in
vacuo without external heating, resulting in an orange solid. The solid
was dissolved in a small amount of MeOH (0.5 mL) and layered with
Et2O (10 mL). After this solution was stored at −20 °C for 16 h, the
complex precipitated as an orange solid. The solution was decanted,
and layering was repeated two more times. The solid was isolated by
scraping and dried in vacuo to provide compound 8 as a
microcrystalline orange solid (29 mg, 38%). Data for 8 are as follows:
mp 154 °C; 1H NMR (400 MHz, CD3OD) δ 8.78 (t, J = 8.8 Hz, 2H),
8.72−8.68 (m, 4H), 8.46−8.43 (m, 1H), 8.18−8.09 (m, 6H), 7.88−
7.83 (m, 3H), 7.80 (t, J = 5.4 Hz, 2H), 7.54−7.45 (m, 5H), 7.32−7.28
(m, 2H), 7.26−7.22 (m, 3H), 5.08 (td, J = 7.4, 2.0 Hz, 1H), 3.51 (t, J =
2.0 Hz, 1H), 3.38 (d, J = 2.0 Hz, 1H), 3.20−3.12 (m, 2H), 3.07−3.02
(m, 1H), 2.98−2.93 (m, 1H), 2.85 (s, 3H), 2.83 (s, 3H), 1.75−1.68
(m, 2H); IR (thin film) 3379, 3229, 3063, 2934, 1638, 1601, 1535,
1462, 1443, 1304, 1271, 1242, 1159, 893, 764, 729 cm−1; HRMS
(ESMS) calcd for C49H46N10O5Ru [M]2+ 479.1433, found 479.1399;
UV−vis λmax 449 nm (ε = 8200 M−1 cm−1), λem 631 nm. Anal. Calcd
for C49H48Cl2N10O5Ru (8·7H2O): C, 50.95; H, 5.41; N, 12.13. Found:
C, 50.66; H, 5.31; N, 11.84.
100, and DTT 8 mM at 25 °C as described previously.34 Cathepsin S
inhibition data were collected using CTSS (4 nM), Z-Val-Val-Arg-
AMC (10 μM), 1, 2, 7, and 8 (1−20 μM) in 50 mM phosphate buffer,
pH 6.5, <1% DMSO, 2.5 mM EDTA, and DTT 8 mM at 25 °C.
Cathepsin B inhibition data were collected using CTSB (4 nM), Z-
Arg-Arg-AMC (10 μM), 1, 2, 7, and 8 (1 nM to 100 μM) in 0.4 M
acetate buffer, pH 5.5, <1% DMSO, 5 mM EDTA, 0.01% Triton X-
100, and DTT 10 mM at 25 °C. CA-074 was used as a positive control
for CTSB inhibition. Data are averages of three independent
experiments with errors equal to standard deviations. Progress curve
data were analyzed using the program Dynafit39 to obtain kinact and Ki
values using a literature method.34
Papain Rhenium Complex MS Analysis. A concentrated
solution of papain (from papaya latex, Sigma-Aldrich, P4762) was
prepared in 20% DMSO/H2O (20 mg/mL), resulting in a suspension
that was filtered through a 0.2 μM membrane. The filtrate (50 μL) was
added to a solution of L-cysteine hydrochloride (2 mg/mL, 200 μL)
and inhibitor 7 (2 mg/mL, 200 μL) in the dark. The solution was
incubated at room temperature for 18 h in the dark and was
concentrated to a volume of ∼50 μL using a 10 kDa centrifugal filter
(Amicon Ultra Ultracel 10K membrane) at 4 °C and 14000g for 30
min. The sample was diluted in 0.2% AcOH/H2O (400 μL) and
concentrated to ∼50 μL. This cycle was repeated four times to remove
excess 7 and L-cysteine. The sample was diluted to a final volume of
450 μL in 0.2% AcOH/H2O and injected (10 μL) into a Waters LCT
Premier XE Micromass KD128 time-of-flight mass spectrometer with
an electrospray source. Spectra were collected from m/z 1000 to 2000,
and the signal was deconvoluted using Masslynx.
Lysate Studies. Lysates obtained from DU-145 and ARCaP(M)
prostate cancer cells were diluted 4× with activating buffer (0.4 M
acetate buffer, 4 mM EDTA, pH 5.5, DTT 8 mM) and incubated for
20 min at room temperature. The substrate Z-Phe-Arg-AMC (100
μM) in assay buffer was added, and the fluorescence of hydrolyzed
AMC was recorded as a function of time. Lysates were incubated with
5−30 μM [Re(phen)(CO)3(1)](OTf) (7) for 15 min and then loaded
directly onto the gel or heated for 5 min at 100 °C and analyzed by
SDS PAGE gel analysis using a Criterion TGX instrument with 4−
20% SDS gradient gel with and 302 nm excitation on a UV
transilluminator.
Cell Studies. The viability effects of 7 on DU-145 cells were tested
via the MTT assay according to the manufacturer’s instructions
(Invitrogen, Waltham, MA). Briefly, DU-145 cells were plated in 96-
well plates (Invitrogen/Thermo Fisher Scientific, Waltham, MA) at 5
× 103 cells per well in 100 μL of DMEM containing 10% FBS. One
plate was seeded for “UV” conditions and one plate for “dark”
conditions. After 24 h of growth, cells were treated in the dark with an
increasing concentration of 7 (supplied in 100 μL of media with the
final DMSO concentration kept at 0.5%), and eight biological
replicates for each concentration were tested per plate. The plates
were loosely wrapped with aluminum foil and placed back in the
incubator for a 2 h incubation at 37 °C, after which both plates were
removed and set at room temperature for 40 min while the “light”
plate was unwrapped and exposed to UV light. The lamp used was a 4
W long-wave UV (365 nm) light, powered at 115 V (UVP, Upland,
CA). Following irradiation, the cells were placed back in the incubator
and kept in the dark under a 5% CO2 atmosphere at 37 °C for another
48 h. Following the required incubation, the medium was removed and
100 μL of fresh medium per well was added followed by the addition
of 10 μL of a 12 mM MTT stock solution (5 mg of MTT dissolved in
1 mL of sterile PBS). A negative control consisting of 100 μL of
medium plus 10 μL of MTT solution was added to empty wells. The
dishes were rewrapped and placed back into the incubator for 4 h.
Medium solution (85 μL) was then removed from each well, and 50
μL of DMSO was added, followed by rocking the plate to ensure
thorough mixing. Cells were incubated at 37 °C for another 10 min
before reading the absorbance at 540 nm on a plate reader.
Lifetime Measurements. The 77 K emission lifetimes were
determined using a Spectra Physics VSL-337ND-S nitrogen laser-
pumped DUO-210 Dye laser system, Hamamatsu P9220 PMT/E717-
63 mounted on a Jobin-Yvon H-100 spectrometer with a National
Instruments NI PCI-5154, 2 GS/s, 1 GHz Digitizer w/8 MB/ch
onboard memory PC card as described previously.38 The laser had
about a 5 ns pulse width, and the instrument response to scattered
excitation light was experimentally determined to have a 1/e decay
time of 9
2 ns. Aqueous sample solutions contained 50 mM
potassium phosphate buffer (pH 7.4) and 10% DMSO.
Enzyme Inhibition. Progress curves for release of free 7-amino-4-
methylcoumarin (AMC) by CTSL were collected using CTSL (4 nM),
Z-Phe-Arg-AMC (10 μM), 1, 2, 7, and 8 (0.05−2.5 μM) in 0.4 M
acetate buffer, pH 5.5, <1% DMSO, 4 mM EDTA, 0.01% Triton X-
Confocal Analysis of Rhenium Compounds in Cells. To
measure the uptake of the rhenium compound by DU-145 cells, we
applied live imaging techniques. Cells were plated in 35 mm dishes
(200000/dish) in DMEM containing 10% FBS and kept in a 37 °C
C
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humidified incubator ventilated with 5% CO2, in line with
recommended growth conditions for these cells. After 48 h of growth,
cells were treated with 30 μM of the Re(I) compound 7 or with
vehicle (DMSO, blank). The DMSO concentration was kept at 0.1%
of the total volume. Cells were incubated for 2 h and then removed
and gently washed three times with PBS. Cells were kept in PBS for
the duration of confocal imaging. Briefly, images of cells were captured
using a Zeiss LSM 780 confocal microscope (Carl Zeiss AG,
Scheme 2. Synthesis of Luminescent Re(I)-Based (A) and
Ru(II)-Based (B) Epoxysuccinyl Inhibitors
Gottingen, Germany) using both 20× and 40× water immersion
̈
lenses. Excitation was set at 405 nm using a diode laser, with the
dichroic filter set at 405/505 nm. All emission from 501 to 740 nm
was collected. Z-stack images (1.5 μm optical slices) of the cells were
taken to capture the complete fluorescence available as opposed to
being limited to a single plane. Four biological replicates per condition
were imaged at 20× to measure the integrated intensity. Intensities of
the images were measured via Volocity software (PerkinElmer,
Waltham, MA), and the integrated intensity per image was quantified
by dividing the total sum of the fluorescence signal (thresholded using
intensity) by the number of cells per image (counted manually). 3D
reconstructed images showing the fluorescence were constructed using
Volocity software.
Assessment of the Effect of Cathepsin L Inhibition on
Rhenium Compound Uptake. We followed a similar procedure to
measure the inhibitory effect of CTSL inhibitor CLIK-148 on the
uptake of the rhenium compound by DU-145 cells. Cells were plated
in 35 mm dishes (150000/dish) under normal growth conditions.
After 48 h of culture, cells were treated with 50 μM of CLIK-148 or
with vehicle (0.5%, DMSO, blank). Cells were incubated with the
inhibitor for 24 h, and then the medium was removed and cells were
gently washed three times with PBS. Cells were then treated with 30
μM of the rhenium compound or vehicle (0.3%, DMSO) and
incubated with the compound for 2 h. Before confocal analysis, the
medium was removed and cells were gently washed three times with
PBS and maintained in PBS for the duration of the imaging protocol.
Confocal analysis was performed as described above for uptake studies.
chloronicotinate (3; Scheme 1A). 2-Pyridylzinc bromide and
methyl 6-chloronicotinate were coupled using a modified
Negishi cross-coupling protocol to obtain the desired
intermediate methyl [2,2′-bipyridine]-4-carboxylate (4).35
Compound 4 was converted to an amide upon treatment
with excess propane-1,3-diamine in refluxing methanol to form
5 in good yield (83%). To complete the synthesis of 2, the
advanced intermediate 6,34 available in 8 steps from D-tartaric
acid (Scheme 1B), was treated with p-nitrophenol and
dicyclohexylcarbodimide (DCC), resulting in the formation of
an activated p-nitrophenyl ester that was combined with 5 to
give 2 in 59% yield over the two steps.
RESULTS AND DISCUSSION
■
Epoxysuccinyl-based inhibitors 1 and 2 were synthesized to
create Re(I) and Ru(II) conjugates. Compound 1 was obtained
following a literature protocol in 10 steps total from D-tartaric
acid.34,40,41 Inhibitor 2 was prepared by starting from methyl 6-
Syntheses of the Re(I)- and Ru(II)-based inhibitors are
shown in parts A and B, respectively, of Scheme 2. The CLIK
analogue 1 was treated with Re(phen)(CO)3OTf36 in CHCl3/
MeOH, giving the Re(I) conjugate 7 in 50% yield after
precipitation from MeOH/Et2O. Heating inhibitor 2 in the
presence of cis-[Ru(bpy)2Cl2]37 in EtOH for 16 h, followed by
layering in MeOH/Et2O, gave Ru(II) conjugate 8 in 38% yield.
Spectroscopic Characterization. Metal complexes 7 and
Scheme 1. Synthesis of Epoxysuccinyl Inhibitor 2
1
8 were characterized by H NMR, IR, electronic absorbance,
and fluorescence emission spectroscopy, electrospray mass
spectrometry, and elemental analysis. Mass spectra for 7 and 8
show ion clusters with prominent peaks at m/z 861.2 and
479.1, respectively, along with suitable isotopic patterns
consistent with those expected for the monocation [Re(phen)-
(CO)3(1)]+ and dication [Ru(bpy)2(2)]2+ (Figures S6 and S9).
The Re(I) conjugate 7 shows electronic absorption (λmax 275
nm, ε = 33300 M−1 cm−1) and emission (λem 543 nm) spectra
consistent with other compounds containing the Re(phen)-
(CO)3 fragment with bound monodentate pyridines (Figure
2).42 Ru(II) conjugate 8 shows absorption (λmax 449 nm, ε =
8200 M−1 cm−1) and emission (λem 631 nm) bands consistent
43
2+
with the Ru(bpy)3 fragment.
1H NMR and IR spectroscopic analyses were used to gain
insight into the structure of complexes 7 and 8. The H NMR
1
spectrum of Re(I) complex 7 in CD3OD shows two doublets at
8.29 and 7.11 ppm, each integrating for two protons, with a
coupling constant of 6.4 Hz, consistent with the 2- and 3-
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Figure 2. Electronic absorption spectra (A) and emission spectra (B) for 7 and 8. Absorption spectra of 7 (blue) and 8 (red). Emission spectra of 7
(black) and 8 (green) were normalized and uncorrected.
Figure 3. Emission decay of 7 and 8 at concentrations of 47.5 and 4.09 μM, respectively, at ambient temperature and 77 K (frozen solution) in
potassium phosphate buffer 50 mM, pH 7.4, and 10% DMSO solvent using 337 nm pulsed excitation monitored at 543 and 631 nm: (A) 7 at 77 K;
(B) 8 at 77 K; (C) 7 at ambient temperature; (D) 8 at ambient temperature. The monoexponential fit (black) and the extracted data (pink) are
indistinguishable; residuals are shown in red. The samples were probably microcrystalline at 77 K. A fast decay component with about 10% of the
substrate amplitude was probably mostly due to the instrument response to scattered excitation light, but it could also contain contribution from
heterogeneities or impurities. Exponential decay fittings were performed in Origin; IRF = 12 ns.
a
Table 1. Kinetic and Thermodynamic Parameters for Inhibition of Cathepsins L, S, and B by 1, 2, 7, and 8
CTSL
CTSS
CTSB
kinact (10−3 kinact/Ki (103 M−1
kinact (10−3 kinact/Ki (103 M−1
kinact (10−3 kinact/Ki (103 M−1
compd
Ki (μM)
s−1
)
s−1
)
Ki (μM)
105
s−1
)
s−1
)
Ki (μM)
s−1
)
s−1
)
b
b
b
1
2
7
8
0.77 0.27
0.74 0.05
0.21 0.04
1.7 0.4
11
5
13 2.8
42.3 4.1
211 42
8.5 2.5
1
31
8
0.29 0.08
5.3 0.2
1933 1573
2.1 0.2
2.7 0.4
0.2 0.2
ND
0.001
0.009
0.001
ND
31
43
14
2
5
1
3.0 0.8
9.4 4.9
3.3 0.9
16 1.3
8.9 4.6
296
8
0.95 0.01
5.5 0.23
194 23
ND
18
3
a
CSTL inhibition data were collected using CSTL (4 nM), Z-Phe-Arg-AMC (10 μM), 1, 2, 7, and 8 (0.05−2.5 μM) in 0.4 M acetate buffer, pH 5.5,
<1% DMSO, 4 mM EDTA, 0.01% Triton X-100, DTT 8 mM at 25 °C. CSTS inhibition data were collected using CSTS (4 nM), Z-Val-Val-Arg-
AMC (10 μM), 1, 2, 7, and 8 (1−20 μM) in 50 mM phosphate buffer, pH 6.5, <1% DMSO, 2.5 mM EDTA, DTT 8 mM at 25 °C. CSTB inhibition
data were collected using CSTB (4 nM), Z-Arg-Arg-AMC (10 μM), 1, 2, 7, and 8 (1 nM to 100 μM) in 0.4 M acetate buffer, pH 5.5, <1% DMSO, 5
mM EDTA, 0.01% Triton X-100, DTT 10 mM at 25 °C. CA-074 was used as a positive control for cathepsin B inhibition. Data are averages of three
b
independent experiments with errors equal to standard deviations. Data are from ref 34.
E
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Figure 4. Model for competitive, irreversible inactivation of cathepsin
L by epoxysuccinyl inhibitors with reversible formation of the enzyme
inhibitor complex (EI) and irreversible formation of the covalent
complex (EI*).
Figure 6. Preparation of a papain-7 conjugate and characterization by
ESMS revealing that the Re(I)(phen)(CO)3 metal fragment remains
intact upon covalent modification of papain by 7.
Figure 5. Re(I)- and Ru(II)-based inhibitors of CTSL 7−10 with
second-order rate constants (kinact/Ki) for irreversible enzyme
inactivation. Data reveal that the nature of the metal complex and
position of attachment strongly influence enzyme inhibition. Data for
9 and 10 are from ref 34.
pyridyl C−H groups of the pyridine ring. A triplet is observed
at 5.06 ppm integrating for one proton consistent with the α-
proton of the phenylalanine residue. In addition, two doublets
with coupling constants of 1.5 Hz were observed at 3.25 and
3.14 ppm, each integrating for one proton, and are consistent
with methine protons of the epoxide. Finally, doublets of
doublets at 3.04 and 2.94 ppm, integrating for one proton each,
were assigned as diastereotopic β-protons of the phenylalanine
residue in 7. Similar resonances were observed in the 1H NMR
spectrum of Ru(II) conjugate 8, including a triplet of doublets
at 5.08 ppm for the α-proton of the phenylalanine residue and
one triplet and one doublet at 3.51 and 3.38 ppm, respectively,
corresponding to the methine protons of the epoxide. The
triplet at 3.51 ppm has a coupling constant of 2 Hz and is likely
Figure 7. Model of CSTL adapted from PDB 3OF8 showing location
of key residues in the S3 subsite.51
the result of merging of two doublets with coupling constants
of ∼2 Hz; one coupling constant is attributed to an adjacent
methine proton and the other 2 Hz coupling constant is
presumably 5J coupling with a disatereotopic CH proton
adjacent to NH. The IR spectrum of 7 demonstrates νCO
stretches at 2029 and 1906 cm−1, indicating the presence of the
fac-[Re(CO)3] core.44
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Lifetime Measurements. Emission lifetimes for 7 and 8 at
concentrations of 47.5 and 4.09 μM, respectively, were
determined in PBS buffer (pH 7.4, 10% DMSO) at 77 K
(frozen solution) and under ambient conditions (Figure 3).
Complexes 7 and 8 were monitored for emission at 543 and
631 nm, respectively, following irradiation at 337 nm (pulse
width 5 ns). The monoexponential fits (black) and original
signal (blue) are indistinguishable; residuals are shown in red.
Lifetimes determined for 7 were 9.50 0.2 μs at 77 K and 0.60
0.02 μs at 293 2 K. Shorter lifetimes were observed for
Ru(II)-based 8: 0.90 0.05 μs at 77 K and 0.06 0.02 μs at
293
2 K. The monoexponential fits (black) and original
signal (blue) are indistinguishable; residuals are shown in red
(Figure 3). These data confirm that 7 and 8 have excited state
lifetimes significantly longer than those of endogenous
fluorophores that contribute to autofluorescence (<10 ns),
giving the Re and Ru conjugates potential for use in time-gated
imaging experiments.30
Figure 8. Viability of DU145 cells in response to compound 7. The
cell viability was determined by MTT assay after 48 h and is reported
relative to control with only the vehicle (0.5% DMSO) added. Cells
were incubated in the presence of 7 (1 nM to 100 μM) at 37 °C for 2
h, followed by 40 min incubation in the dark at room temperature or
irradiated with a 4 W long-wave UV (365 nm) light, powered at 115 V.
Error bars represent the standard error of the mean of eight replicate
wells, and data are representative of three independent experiments.
Enzyme Inhibition. Compounds 1, 2, 7, and 8 were
evaluated for inhibition of cysteine cathepsins B (CTSB), L
(CTSL), and S (CTSS) to determine how attachment of the
metal complexes influenced enzyme inhibition. All three
enzymes are endopeptidases that are abundantly expressed in
a variety of human tissues.45 In addition, CTSB also shows
dicarboxypeptidase activity.3,46 Cathepsin inhibition was
measured using progress curve assays with continuous
monitoring of fluorogenic substrate hydrolysis. Solutions of
aminomethylcoumarin (AMC)-based fluorogenic substrates
(10 μM) and inhibitor (0.05−20 μM) in 50 mM phosphate
or 0.4 M acetate buffer (see Table 1 for exact conditions) were
treated with the enzyme CTSL, CTSS, or CTSB (4 nM)
activated with DTT (8 mM) and were immediately analyzed
for formation of free AMC over the course of 15 min. Data
Figure 9. Confocal images of live DU-145 cells treated with 7 (30
μM): fluorescence (left), bright field microscopy (middle), and overlay
(right).
Figure 10. Uptake of 7 by living prostate cancer cells: (A−D) Volocity export images of untreated (blank; A, C) and 7-treated (30 μM, B, D) DU-
145 cells; (E) Quantification of intracellular fluorescence intensity upon compound 7 treatment; (F−H) reduction of intracellular fluorescence with
cathepsin L inhibition by pretreatment with CLIK-148 (50 μM) for 24 h before 7 (30 μM).
G
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were fit to a standard two-step model for irreversible enzyme
inhibition, where noncovalent binding of the inhibitor and
irreversible covalent bond formation to form the inactivated
enzyme EI* compete with hydrolysis of substrate S to form
product P (Figure 4). By following substrate hydrolysis over
time in the presence of variable amounts of inhibitor, the
thermodynamics of reversible inhibitor binding (Ki) and the
rate constant for irreversible covalent bond formation with the
inhibitor (kinact) can be obtained. The quotient kinact/Ki is an
important parameter that is often used as a measure of potency
and represents the second-order rate constant for enzyme
inactivation by the irreversible inhibitor.
Mechanism of Enzyme Inhibition. Literature data reveal
that rhenium compounds, specifically Re(V)−oxo complexes,
are potent inhibitors of cysteine proteases.27 In these cases, the
rhenium metal center undergoes direct ligation to the
nucleophilic thiolate in the cysteine protease active site. To
gain further insight into the mode of inhibition of CTSL by 7,
several more experiments were carried out. The complex
[Re(phen)(CO)3(pyridine)](OTf)49 was used as a control to
probe for potential rhenium binding but showed no inhibition
of CTSL at concentrations up to 100 μM, in stark contrast with
7, which inhibits CTSL in the low-nanomolar range. Binding of
7 to the cysteine protease papain, a close model of CTSL,50 was
probed by MS analysis. Treatment of papain with excess 7,
followed by isolation through a size exclusion filter and ESMS
analysis, revealed a major product with MW = 24283 (Figures
S24 and S25), consistent with the calculated mass for the
papain-1-[Re(phen)(CO)3] adduct (Figure 6). These data are
consistent with the epoxysuccinyl group, rather than the Re(I)
metal center, forming a covalent bond with inhibitor 7 through
attack of the active site thiolate on the epoxide, resulting in ring
opening. Taken together, these data indicate that the Re
complex of 7 remains intact and does not undergo ligand
substitution due to direct binding to the thiolate. Instead, the
enhancement of inhibition observed between 1 and 7 is likely
due to favorable noncovalent interactions between the cationic
Re(I) metal complex and CTSL, likely a carboxylate-rich area
near the S3 subsite (Figure 7).
Cellular Studies. Rhenium conjugates of biologically active
molecules have shown great success in cell imaging experi-
ments.29,30,33,52−61 To probe whether compound 7 would be
appropriate for cell imaging experiments, we used DU-145
cancer cells, an aggressive form of prostate cancer cells that are
known to express high levels of CTSL in culture.62 First, we
assessed the effects of 7 on cell viability. Re(I) complex 7 was
well tolerated by the DU-145 cells, with only minor toxicity
observed, except at the highest concentration surveyed (100
μM) under light (365 nm irradiation) or dark conditions
(Figure 8), as judged by the MTT assay after 48 h.
Next, DU-145 cells were treated with 7 (30 μM) for 2 h
under regular growth conditions and then washed with PBS
buffer and imaged by confocal microscopy, using 405 nm
excitation. Compound 7 clearly caused a significant increase in
intracellular fluorescence intensity (Figure 9). To probe for
CTSL binding, DU-145 cells were treated with the CTSL-
selective inhibitor CLIK-148 (50 μM) for 24 h prior to treating
with 7 (30 μM). Intracellular fluorescence intensity decreased
significantly by over 50%, consistent with CLIK-148 and 7
competing for the same binding sites (Figure 10). These data
are consistent with 7 being nontoxic, cell-permeable, and able
to label active CTSL in living prostate cancer cells.
In order to further probe for the selectivity of CTSL binding,
Re(I) compound 7 was evaluated by SDS-PAGE analysis of cell
lysates from DU-145 and ARCaP(M) prostate cancer cells,
which both express high levels of CTSL. First, the lysates were
prepared and assayed for activity of CTSL using progressive
curves for hydrolysis of Z-Phe-Arg-AMC. Fluorescence read-
ings increased linearly 2.6-fold in PC3 and 3-fold in ARCaP(M)
over the course of 15 min, demonstrating that CTSL is active in
both cell lysates. With lysates containing catalytically active
CTSL in hand, samples were incubated with 5 μM [Re(phen)-
(CO)3(1)](OTf) (7) for 15 min and then heated for 5 min at
100 °C and analyzed by SDS PAGE gel analysis. Unfortunately,
we unable to detect a fluorescent band corresponding to the
Inhibition data for compounds 1, 2, 7, and 8 of CTSL, CTSS,
and CTSB are presented in Table 1. Inhibitor 1 gave kinact/Ki =
13000 2800 M−1 s−1, which is within experimental error of
the value for the potent CTSL inhibitor CLIK-148 (11000
3000 M−1 s−1).47 The bipyidine derivative 2 showed Ki = 0.74
0.05 μM, similar to that of 1, but demonstrated a significantly
higher rate of irreversible inactivation (kinact = 0.031 0.002
s−1) in comparison to 1, which gave a kinact/Ki value for 2 for
CTSL inactivation roughly 3 times that of 1. Both compounds
1 and 2 were selective for CTSL over the related cysteine
protease CTSS, with kinact/Ki ratios for CTSL/CTSS of 45 and
8, respectively. Neither 1 nor 2 inhibited CTSB potently, with
k
inact/Ki values of around 1.00 M−1 s−1. Interestingly, compound
7, the Re(phen)(CO)3 conjugate of 1, was much more potent
than 1 and 2, with a second-order rate constant of 211000
42000 M−1 s−1, greater than 16 times that for 1. This makes 7
among the most potent irreversible CTSL inhibitors known to
date.18,48 Data revealed that the higher potency for 7 vs 1 was
due to tighter reversible binding (∼4-fold) and faster
inactivation (∼4-fold). In addition, 7 showed an enhanced
selectivity over 1 for CTSL vs CTSS inhibition, increasing the
CTSL/CTSS kinact/Ki ratio from 45 with 1 to 222 for 7. In
other words, attaching the Re(phen)(CO)3 unit to 1 improved
both potency and selectivity for inhibition of CTSL over CTSS.
Like 1 and 2, Re(I) compound 7 was not a potent inhibitor of
CTSB. Ru(II)-based compound 8 showed behavior very
different from 7. First, 8 was roughly 25 times less potent
against CTSL than 7, with a kinact/Ki value of only 8500 2500
M
−1 s−1. Second, attaching the Ru(bpy)2 fragment to 2 to give
8 actually decreased rather than increased the kinact/Ki value for
8 relative to 2 by a factor of almost 5. Third, the selectivity for
CTSL over CTSS was eroded for 8, with a ratio of only 1.5, in
comparison to 222 for 7.
Taken together with data from previously synthesized
metal−inhibitor conjugates (Figure 5),34 our results collectively
confirm that the position and nature of the metal center
strongly influence potency and selectivity of CTSL inhibition. A
comparison of conjugates 7 and 9, which contain the same
parent inhibitor structure but different metal fragments,
Re(phen)(CO)3 and Ru(tpy)(Me2bpy), respectively, indicates
that the Re(I) group is much better tolerated than the more
bulky Ru(II) fragment in the CTSL active site. Compounds 8
and 9 have Ru(II) fragments with similar size but different
linker lengths between the epoxysuccinyl group and the metal
center and differ by a factor of less than 2 for kinact/Ki values,
suggesting that hydrophobic cationic groups are tolerated
reasonably well by CTSL. In contrast, placing Ru(tpy)-
(Me2bpy) on the pyridylalanine side chain of 10 strongly
decreases the rate of inhibitor inactivation, with a kinact/Ki value
of only 570 60 M−1 s−1.
H
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to new frontiers. Biochim. Biophys. Acta, Proteins Proteomics 2012, 1824
(1), 68−88.
labeled CTSL (37 kDa) or any other larger proteins by gel
analysis. Instead, we detected a fluorescent band moving at the
dye front (<8 kDa). Similar experiments at higher concen-
trations of 7 (30 μM) with and without boiling the sample
before analysis were conducted but gave the same results. We
considered that the covalent cathepsin L-7 adduct might not be
stable to gel analysis, presumably because it is electrochemically
active, and labile and is released from the monopyridine donor
upon subjection to SDS-PAGE analysis. Even though our data
confirmed that 7 is a highly potent, selective, and irreversible
inhibitor of CTSL that is cell permeable, further experiments
will be needed to confirm the selectivity of 7 for labeling CTSL
in the presence of other proteins, as present in the complex
milieu of lysates and whole cells.
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CONCLUSION
■
In conclusion, we report a Re(I)-based inhibitor of CTSL that
is highly potent, selective, and cell permeable. Our data are
consistent with the Re(I) complex enhancing binding to CTSL
and increasing the rate of irreversible alkylation, making 7
among the most potent inhibitors of CTSL recorded to date.
Importantly, this study reveals a new strategy for using
coordinatively saturated metal complexes to enhance potency
and selectivity of inhibitors through favorable noncovalent
interactions using metal centers. This strategy is novel and
distinct from using complexes with vacant coordination sites as
enzyme inhibitors to ligate nucleophilic enzyme side chains or
complexes that bind directly inside active sites.63,64 Given that
transition metals such as Re are not native in biology and are
subject to nontraditional methods for quantification and
imaging, such as ICP-MS and IR-based spectromicroscopy, 7
and derivatives thereof may hold promise as reagents for
enzyme inhibition, detection, and quantification.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b00978.
(15) Blum, G.; von Degenfeld, G.; Merchant, M. J.; Blau, H. M.;
Bogyo, M. Noninvasive optical imaging of cysteine protease activity
using fluorescently quenched activity-based probes. Nat. Chem. Biol.
2007, 3 (10), 668−677.
(16) Verdoes, M.; Oresic Bender, K.; Segal, E.; van der Linden, W.
A.; Syed, S.; Withana, N. P.; Sanman, L. E.; Bogyo, M. Improved
Quenched Fluorescent Probe for Imaging of Cysteine Cathepsin
Activity. J. Am. Chem. Soc. 2013, 135 (39), 14726−14730.
(17) Ben-Nun, Y.; Merquiol, E.; Brandis, A.; Turk, B.; Scherz, A.;
Blum, G. Photodynamic quenched cathepsin activity based probes for
cancer detection and macrophage targeted therapy. Theranostics 2015,
5 (8), 847−862.
1H and 13C NMR and HRMS spectra for new
compounds, enzyme inhibition studies, papain rhenium
conjugate and MS analysis, and lifetime measurements
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail for J.J.K.: jkodanko@chem.wayne.edu.
■
ORCID
(18) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Irreversible
inhibitors of serine, cysteine, and threonine proteases. Chem. Rev.
2002, 102 (12), 4639−4750.
John Endicott: 0000-0002-8998-1030
Jeremy J. Kodanko: 0000-0001-5196-7463
Notes
(19) Frizler, M.; Stirnberg, M.; Sisay, M. T.; Guetschow, M.
Development of nitrile-based peptidic inhibitors of cysteine cathepsins.
Curr. Top. Med. Chem. 2010, 10 (3), 294−322.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(20) Katunuma, N.; Murata, E.; Kakegawa, H.; Matsui, A.; Tsuzuki,
H.; Tsuge, H.; Turk, D.; Turk, V.; Fukushima, M.; Tada, Y.; Asao, T.
Structure based development of novel specific inhibitors for cathepsin
L and cathepsin S in vitro and in vivo. FEBS Lett. 1999, 458 (1), 6−10.
(21) Kellett, A.; Prisecaru, A.; Slator, C.; Molphy, Z.; McCann, M.
Metal-Based Antimicrobial Protease Inhibitors. Curr. Med. Chem.
2013, 20 (25), 3134−3151.
■
We gratefully acknowledge the National Institutes of Health
(Grant EB 016062) and Wayne State University for their
generous support of this research.
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