Evaluation of α,β-unsaturated ketone-based probes for papain-family cysteine proteases

Article abstract of DOI:10.1016/j.bmc.2008.02.089

The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, α,β-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.

Full text of DOI:10.1016/j.bmc.2008.02.089

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 1071–1078
Evaluation of a,b-unsaturated ketone-based probes
for papain-family cysteine proteases
a
a,c
Zhimou Yang, Marko Fonovic, Steven H. L. Verhelst,a
´
Galia Bluma and Matthew Bogyoa,b,
*
aDepartment of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
bDepartment of Microbiology and Immunology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA
cDepartment of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
Received 15 December 2007; revised 21 February 2008; accepted 27 February 2008
Available online 2 March 2008
Abstract—The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of
enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target
diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive func-
tional groups for use in ABPs. In this study we evaluate the utility of the, a,b-unsaturated ketone reactive group for use in
ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cys-
teine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended
on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the
potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling
properties.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
zyme.1–5 These probes contain three main elements:
(1) a reactive functional group that facilitates the for-
mation of the covalent bond with the active site cata-
lytic residue of a target (2) a linker that can be used
to control the specificity of binding interactions be-
tween the probe and target enzyme and (3) a tagging
group that allows probe labeled targets to be isolated,
biochemically characterized or imaged. While the past
10 years has seen a significant growth in both the
diversity of ABPs and the types of enzymes that can
be targeted by these probes,6,7 there remains a need
to develop new classes of ABPs in order to continue
to expand the repertoire of proteins that can be stud-
ied using chemical proteomic methods. In particular,
new reactive ‘warheads’ need to be evaluated for use
with general probe scaffolds. These reactive functional
groups often control the broad selectivity of the ABP
and also have a dramatic impact on the overall cross-
reactivity resulting from non-specific interactions with
abundant background proteins.
Most enzymes are regulated by a complex set of controls
that prevent them from causing damage to a cell as the
result of their uncontrolled activation. Proteases are no
exception, as virtually all members of this enzyme class
are regulated by initial activation of a zymogen form
and then subsequent temporal control by endogenous
inhibitors. Thus, methods such as transcript array pro-
filing and standard proteomics are unable to provide
information about the dynamic, post-translational regu-
latory mechanisms used to control the networks of pro-
teolytic events involved in basic cell physiology or
disease pathology. Therefore, it is necessary to develop
tools that allow the activity levels of specific protease
targets to be monitored within the context of a complex
biological environment.
Activity-based probes (ABPs) are small molecules that
form activity dependant covalent bonds to a target en-
While a number of effective reactive functional groups
have been employed in ABPs that target cysteine prote-
ases (for reviews see Refs. 2, 3, 7, and 8), there still re-
mains room for improvement both at the level of
Keywords: Activity-based probes; Cysteine protease; Active site label-
ing; Irreversible inhibitor.
*
Corresponding author. Tel.: +1 650 725 4132; fax: +1 650 725
7424; e-mail: mbogyo@stanford.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.089

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Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
selectivity and also at the level of ease of synthesis. The
peptide vinyl sulfones have found widespread use in
probes that target papain-family cysteine proteases9–11
as well as ubiquitin specific proteases12–14 and the pro-
teasome.15,16 However, this reactive functional group
is not effective for all classes of cysteine proteases. The
highly related Michael acceptor, the a,b-unsaturated ke-
tone has been extensively used in the development of
inhibitors of specific classes of cysteine proteases.17,18
Interestingly, while there is ample evidence to suggest
that the a,b-unsaturated ketone has different specificity
properties relative to the vinyl sulfone, this class of war-
head has only been used in a single class of highly spe-
cific activity-based probes that target the de-
ubiquitinating enzymes (DUBs).19 This class of probe
derives the majority of its selectivity from the large 76
amino-acid ubiquitin protein that is attached to the
reactive warhead group. Thus, it remains unclear how
valuable the a,b-unsaturated ketone is as a more general
warhead for use with short peptide-based ABPs.
2. Results and discussion
2.1. Evaluation of the a,b-unsaturated ketone as a
warhead for use in ABPs
We initially set out to evaluate the utility of the a,b-
unsaturated ketone (abUK) as a reactive group for use
in ABPs that target cysteine proteases. We identified a
related abUK in a small molecule screen for compounds
that block host cell invasion by the obligate intra-cellu-
lar parasite Toxoplasma gondii.21 Based on the structure
of this hit we designed and synthesized a series of ana-
logs that contain a central P2 phenylalanine and P1 ala-
nine peptide attached to the a,b-unsaturated methyl
ketone. The simplest analog, yzm09 (5a) contains a
Cbz cap at its N-terminus. This capping group is re-
placed by biotin (yzm13, 5c) or by a fluorescent tag
(yzm24, 5d) to yield the first generation ABPs. The syn-
thesis of yzm09 was accomplished by preparation of the
dipeptide Z-Phe-Ala (2a) followed by conversion to the
corresponding Weinreb amide (3a); the reduction of the
Weinreb amide and further treatment with the Wittig re-
agent of 1-triphenyl-phosphoranylidene-2-propanone
afforded a mixture of the desired product with trans-
conformation and its cis isomer (Scheme 1). The ratio
of trans/cis was determined to be approximately 4/1
based on HPLC analysis with the ratio being highly
dependant on the steric properties of the P1 sidechain
as has been shown for peptide vinyl sulfones.22 The de-
sired compound with trans-conformation (yzm09, 5a)
was separated by HPLC in reasonable yield (36% for
five steps). The labeled analogs of yzm09 were prepared
from the Boc-protected compound 5b. After deprotec-
tion of the Boc-group, biotin or BODIPY-TMR-X
was used to generate the corresponding labeled probes
(yzm13 and yzm24, respectively, in Scheme 2).
In this study we synthesized a number of simple peptide
a,b-unsaturated ketones and tested them as general
activity-based probes of the cysteine protease family.
We find that biotinylated and fluorescent a,b-unsatu-
rated ketones show very potent labeling of cathepsins
with low overall background labeling in total tissue
and cell extracts, respectively. Compared with the previ-
ously validated acyloxymethyl ketone (AOMK)-based
probes, the a,b-unsaturated ketone showed enhanced
labeling in tissue and cell extracts and similar levels of
labeling in intact cells. Importantly, the a,b-unsaturated
ketones were several orders of magnitude more potent
toward the recombinant cathepsins B and L compared
to a related AOMK, suggesting that they may provide
higher levels of signal for in vivo imaging using the
methods outlined for the AOMK-based ABPs.20 Fur-
thermore the a,b-unsaturated ketones can be readily
synthesized from the corresponding peptide Weinreb
amides and can therefore be used to make diverse pep-
tide sequences for evaluations of new classes of specific
ABPs.
2.2. Evaluation of the biotinylated ABP yzm13
Having successfully synthesized two labeled analogs of
the parent lead compound yzm09, we began testing the
biotin probe yzm13 for labeling of target proteases in
O
O
H
H
i, ii
iii
R
OH
R
N
R
N
O
N
N
OH
N
N
H
H
H
O
O
O
2a R=Cbz
2b R=Boc
1a R=Cbz
1b R=Boc
3a R=Cbz
3b R=Boc
O
O
iv
v
H
H
R
N
R
N
N
N
H
H
O
O
5a R=Cbz
5b R=Boc
4a R=Cbz
4b R=Boc
Scheme 1. Synthetic scheme for yzm09 and 5b. Reagents: (i) DCC, NHS, CHCl3; (ii) L-alanine, NaHCO3, acetone/H2O; (iii) HBTU, DIPEA, N,O-
dimethyl-hydroxylamine hydrochloride, DMF; (iv) LiAlH4, THF; (v) 1-triphenyl-phosphoranylidene-2-propanone, DCM.

Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
1073
O
O
O
H
N
ii
i
H
N
O
N
H
H2N
O
O
O
5b
6
O
O
O
O
NH
H
H
N
O
O
N
H
O
N
H
HN
H
S
O
yzm13
O
O
O
H
N
N
H
N
H
or
N
O
O
N
B
F
yzm24
F
O
O
H
N
O
N
H
O
yzm09
Scheme 2. Synthetic scheme for yzm13 and yzm24. Reagents: (i) 4 M HCl in dioxane; (ii) DIPEA, acetone.
complex proteomes. We initially used yzm13 in crude
mouse tissue extracts derived from liver, spleen, heart,
kidney, ovary, and uterus. Total protein extracts at pH
5.5 were pre-incubated with DMSO vehicle or the unla-
beled parent compound yzm09 followed by the addition
of the biotin probe yzm13 at a range of probe concentra-
tions. Labeled proteins were analyzed by SDS–PAGE
followed by detection using HRP-coupled streptavidin
(Fig. 1). These initial labeling results confirmed that
the biotin probe yzm13 strongly labeled one predomi-
nant protein just below the 36 kDa molecular weight
marker. The labeling of this predominant protein was
completely blocked by pre-treatment of the samples with
10 lM concentrations of yzm09 suggesting that it was
the only selectively labeled protein in the extract. While
a number of higher molecular weight species were ob-
served at high concentrations of the probe, overall
yzm13 showed highly specific labeling of a single target
that could be observed at probe concentrations as low
as 10 nM. Thus, the abUK is a potentially valuable war-
head for use in ABPs as it shows effective labeling at
very low concentrations of the probe.
We next wanted to identify the predominant labeled
protease observed with yzm13 in mouse tissue extracts.
We therefore performed an affinity purification experi-
ment in which mouse liver lysates with or without the
pre-treatment of 10 lM of yzm09 were labeled with
1.0 lM of yzm13. After enrichment of biotin labeled
proteins on immobilized avidin, both samples were pro-
cessed by ‘on bead’ trypsin digestion to release peptides
for subsequent analysis by LC–MS/MS.23 Analysis of all
isolated peptides confirmed the presence of cathepsin B
(Cat B) as well as several background proteins including
endogenously biotinylated proteins such as propionyl-
coenzyme A carboxylase and abundantly expressed pro-
teins such as hemoglobin and transferases. Importantly,
of all the nine proteins identified, only cathepsin B
Figure 1. Labeling of mouse tissue lysates with the biotin-labeled probe yzm13. Total crude extracts (pH 5.5) from the indicated mouse tissues were
normalized for total protein and labeled by addition of yzm13 at the final concentrations indicated. In some samples the unlabeled control inhibitor
yzm09 was added prior to addition of the probe. Samples were resolved by SDS–PAGE and labeled proteins visualized by affinity blotting using
streptavidin–HRP as outlined in Section 4.

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Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
labeling of Cat B with very low overall background
when the probe was used at 500 nM final concentra-
tions, consistent with the observed labeling pattern of
the biotin labeled probe yzm13. When yzm24 was used
to label total extracts from a human breast cancer cell
lysate, again a band of labeled protein corresponding
to cathepsin B was observed (Fig. 2B). While these cell
extracts showed a significantly higher level of back-
ground labeling compared to the spleen lysates, only
labeling of the specific cathepsin B band was abolished
by pre-treatment of the sample with yzm09. Thus, the
fluorescently labeled yzm24 shows similar specific label-
ing patterns as the biotin probe yzm13. Both reagents
are highly specific labels of cathepsin B and can be used
at sub-micromolar concentrations.
Figure 2. Labeling of total cell extracts with the fluorescently labeled
probe yzm24. (A) Crude protein extracts from mouse spleen tissue (pH
5.5) was labeled by addition of yzm24 at the indicated final concen-
tration followed by SDS–PAGE and analysis of labeled proteins by
scanning of the gel for fluorescence using a flatbed laser scanner. The
unlabeled inhibitor yzm09 was used for pre-treatment of the sample to
identify specific labeled proteins. (B) MDA-MB231 cell lysates were
labeled with yzm24 at the indicated concentration and analyzed as
outlined in (A).
2.4. Labeling in intact cells with the cell permeable
fluorescent probe yzm24
As a final test of the abUK probe, we wanted to test the
utility of this probe class for labeling of endogenous cys-
teine proteases in intact cells. Since the biotin labeled
probe yzm13 was unlikely to penetrate the cells and gain
access to intra-lysosomal pools of cathepsins we decided
to focus our attention on the BODIPY labeled analog
yzm24. Our group has previously shown that the BOD-
IPY labeled version of the AOMK probe GB111 is use-
ful for intact cell labeling experiments.24 Thus, we
compared the labeling of intact mouse fibroblast cells
by the abUK containing fluorescent probe yzm24 to
labeling by the related AOMK probe GB111. In wild
type fibroblasts we observed the previously reported
characteristic labeling pattern of GB111 that includes
a single form of cathepsin B (32 kDa) and the heavy
showed a significant decrease in the number of peptides
identified in the sample that had been pre-treated with
yzm09 to block specific labeling of targets. This result
demonstrated that the only specific target of yzm13 in
total liver extracts is Cat B, consistent with the labeling
observed by SDS–PAGE analysis.
2.3. Labeling complex proteomes with the fluorescent
ABP yzm24
To confirm that the abUK could also be used with fluo-
rescent detection, we tested the ability of yzm24 to label
mouse tissue lysate and also cells lysate. The fluores-
cently labeled yzm24 strongly labeled Cat B, as indicated
by the intense band just above 28 kDa in mouse spleen
lysate that was completely blocked by pre-treatment
with yzm09 (Fig. 2A). Again, we observed highly specific
and light chain forms of cathepsin
L (28 and
22 kDa).24 We observed a highly similar labeling pattern
for yzm24 but with a higher level of background labeling
(Fig. 3A). To confirm that yzm24 was labeling both
cathepsin B and cathepsin L as observed for GB111,
Figure 3. Labeling of intact cells with GB111 and yzm24. (A) Intact wildtype, cathepsin B deficient (Cat Bꢀ/ꢀ) or cathepsin L deficient (Cat Lꢀ/ꢀ
)
fibroblasts were labeled by addition of the fluorescent probes GB111 and yzm24 to the culture media at the concentrations indicated. For some
samples, the unlabeled inhibitor yzm09 was used to pre-treat cells for 30 minutes prior to labeling with the probe. Cells were lysed by addition of SDS
sample buffer and samples were analyzed by SDS–PAGE followed by scanning of the gels for fluorescence using a flatbed laser scanner. (B) Intact
NIH-3T3 cells were pre-treated with DMSO vehicle, yzm09 or the general papain-family protease inhibitor JPM-OEt and then labeled with GB111 or
yzm24 as in (A). The location of cathepsins B and L is indicated.

Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
1075
Table 1. Inhibition rate constants of yzm09 for human cathepsin B
and cathepsin L
mine if they represent useful tools for in vivo imaging
studies.
Enzyme
Cat B (Mꢀ1 sꢀ1
36,680 3,952
)
Cat L (Mꢀ1 sꢀ1
1,634,400 75,260
)
yzm09
4. Experimental
4.1. General
we labeled extracts for mouse fibroblasts derived from
the cathepsin B and cathepsin L knock out mice (Cat
Bꢀ/ꢀ and Cat Lꢀ/ꢀ, respectively; Fig. 3A). As expected,
the 32 kDa labeled protein disappeared in Cat Bꢀ/ꢀ cells
and the lower two bands disappeared in Cat Lꢀ/ꢀ cells,
thus confirming that yzm24 efficiently labels both
cathepsin B and cathepsin L in intact cells. This result
was initially somewhat surprising considering the overall
highly specific labeling of cathepsin B in both cell and
tissue extracts. However, we and others have found that
cathepsin L activity is lost once cells or tissues are
lysed.25 This may be due to the presence of endogenous
inhibitors that are released upon lysis that quickly inac-
tive the mature active forms of cathepsin L or it may be
due to a relatively poor stability of cathepsin L in con-
ditions used in our buffers.
All chemicals and resin were purchased from commercial
suppliers and used without further purification. All water
sensitive reactions were conducted in anhydrous solvents
under positive pressure of argon. Reactions were ana-
lyzed by LC/MS with an API 150EX single quadrupole
mass spectrometer (Applied Biosystems). Reverse phase
˚
HPLC was used with an AKTA explorer 100 (Amersham
Pharmacia Biotech) with C18 columns. High-resolution
MS analyses were performed by the Stanford Proteomics
and Integrative Research Facility with an Autoflex MAL-
DA TOF/TOF mass spectrometer (Bruker). Fluorescent
gels and plates were scanned with a Typhoon 9400 flatbed
laser scanner (GE Healthcare).
4.2. Synthesis
As a final test of the abUK containing probes, we
wanted to confirm our findings that these compounds
are efficient labels of both cathepsin B and L. We there-
fore measured the overall kinetic rate constants of inhi-
bition of the parent yzm09 for the recombinant forms of
cathepsin B and cathepsin L. The results confirm that
yzm09 shows fast, irreversible inhibition of both cathep-
sins B and L (Table 1). The kass of yzm09 for Cat B and
Cat L are 36,680 and 1,634,400 Mꢀ1 sꢀ1, respectively,
indicating that it is more than 10-fold more potent than
the selective probe GB111.24 Thus, these results suggest
that the increased potency of the abUK containing
inhibitors relative to the AOMK containing probes
may be due to the overall increased reactivity of this
class of warhead.
Cbz-FA-ketone (yzm09) and Boc-FA-ketone (5b). Z-
Phe (299 mg, 1 mmol) and NHS (115 mg, 1 mmol) were
dissolved in 20 mL of chloroform, and DCC (230 mg,
1.1 mmol) was added to the above solution with stirring
at room temperature. After 1 h the formed solid was fil-
tered off and the solvent was removed under reduced
pressure. The crude product was used in the next reac-
tion without further purification.
L-Alanine (89 mg, 1 mmol) and NaHCO3 (168 mg,
2 mmol) were dissolved in 20 mL of water with stirring.
Next, a solution of crude product obtained above (dis-
solved in 40 mL acetone) was added. The resulting reac-
tion mixture was stirred at room temperature overnight.
The reaction mixture was concentrated under reduced
pressure. Subsequently, 30 mL of water was added and
the precipitate was removed by filtration. The filtrate
was acidified to a pH of ꢁ3, resulting in precipitation
of the product. The solid was obtained by filtration,
washed with water, and dried in vacuo (284 mg; 77%
yield). The crude product was used without further
purification.
3. Conclusions
Herein, we described the evaluation of the a,b-unsatu-
rated ketone (abUK) as a warhead for use in activity-
based probes for papain-family cysteine proteases. We
demonstrate that this class of reactive functional group
allows rapid synthesis of ABPs that show excellent label-
ing properties in tissues and cell extracts as well as in in-
tact cells. While the improved potency of this class of
compounds toward the target cathepsins relative to pre-
viously reported AOMK-based probes may be beneficial
in that it allows the use of lower probe concentrations, it
comes with the price of potentially higher levels of back-
ground labeling. Thus, further studies may be required
to determine if alternate peptide scaffolds can be used
to enhance selectivity of the abUK containing probes.
Regardless we believe that this class of compound will
find use for development of ABPs for diverse cysteine
protease families, especially for those targets that are
not efficiently labeled by currently used warheads such
as the AOMK and vinyl sulfone. We are currently eval-
uating the properties of this probe class in vivo to deter-
About 185 mg of the crude material obtained in the pre-
vious step, N,O-dimethyl-hydroxylamine hydrochloride
(47 mg, 0.5 mmol), and HBTU (190 mg, 0.5 mmol) were
dissolved in 5 mL of anhydrous DMF. Next, 194 lL of
DIEA was added. The resulting mixture was stirred at
room temperature for 1 h, after which the solvent was
concentrated under reduced pressure. 100 mL of ethyl
acetate was added and the organic phase was washed
with dilute acid (3· 40 mL) and brine (1· 40 mL), dried
(MgSO4), and concentrated under reduced pressure. The
Weinreb amide 3a was purified by flash column chroma-
tography (17–50% ethyl acetate in hexanes) and ob-
tained as a white solid (162 mg, 78%).
To a solution of Weinreb amide 3a (162 mg) in 10 mL of
anhydrous THF, 34 mg of LiAlH4 was added at 0 ꢁC.

1076
Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
After half an hour, 20 mL of 5% KHSO4 and 50 mL of
ethyl acetate were added. The organic layer was washed
with KHSO4 (2· 20 mL), brine, dried (MgSO4), and
concentrated under reduced pressure. The resulting
aldehyde was obtained as a white solid.
centration). Labeling was carried out for 1 h at room tem-
perature. Samples were separated by SDS–PAGE and
transferred to a nitrocellulose membrane (BioRad,
USA). The membrane was blocked in casein (5% solution
in PBS) and incubated with streptavidin–HRP (dilution
1:3500, Sigma, USA) for 1 h at room temperature. Biotin-
ylated proteins were visualized using SuperSignal West
Pico Chemiluminescent Substrate (Pierce, USA).
To the solution of aldehyde (dried by coevaporation with
toluene) in 15 mL DCM, 1-triphenyl-phosphoranylidene-
2-propanone (160 mg) was added and stirred at room
temperature overnight. The reaction mixture was concen-
trated and the resulting pale yellowish solid was purified
by HPLC. 92 mg of yzm09 was obtained (35.8% overall
yield, based on Z-Phe-OH). 1H NMR (300 MHz, CDCl3):
d 1.16 (d, 3H, CH–CH3), 2.194 (s, 3H, CH3), 2.97–3.02
and 3.10–3.15 (m, 2H, –CH–CH2–Ph), 4.33–4.35 (m,
1H, –CH–CH2–Ph), 4.61–4.64 (m, 1H, CH–CH3), 5.08
(s, 2H, –O–CH2–Ph), 5.86–5.90 (d, 1H, –CH–CH@),
6.41–6.46 (d, 1H, @CH–CO–), and 7.18–7.34 (m, 10H,
aromatic). MS (EI): [M+H]+ Calculated: (394.2); found:
(395.2).
4.4. Affinity enrichment and elution of labeled proteins
After protein labeling, free probe was removed by a PD-
10 gel filtration column (Amersham, USA) and sample
was eluted in PBS buffer (pH 7.4). Streptavidin beads
(Pierce, USA) were washed with PBS buffer and added
to the PD-10 eluate. The sample was incubated at room
temperature with shaking for 1 h. Streptavidin beads
were separated from the unbound fraction by centrifu-
gation. The supernatant was discarded and the beads
were sequentially washed with a series of PBS buffers
containing 0.05% SDS, 1 M NaCl, and 10% EtOH. Fi-
nally, the beads were washed with 100 mM ammonium
hydrogen carbonate. Washing was performed three
times with each buffer solution.
A similar procedure was used to afford the compound 5b
(overall yield = 24.3%, based on Boc-Phe-OH). MS (EI):
[M+H]+ Calculated: (360.2); found: (361.2).
Biotin-FA-ketone (yzm13) and BODIPY-TMRX-FA-
Ketone (yzm24). 100 mg of compound 5b was dissolved
in 6 mL of 4 M HCl in dioxane, the reaction mixture
was stirred at room temperature for 4 h. The organic
solvent was removed under reduced pressure. The ob-
tained viscous liquid was coevaporated with toluene
three times then dissolved in 10 mL of acetone and kept
in the ꢀ20 ꢁC refrigerator.
4.5. Sample preparation for LC–MS/MS analysis
For ‘on bead’ digestion, streptavidin beads with bound
proteins were resuspended in 100 lL of denaturing buffer
(50 mM sodium hydrogen carbonate, 6 M urea). Bound
proteins were reduced in the presence of 10 mM DTT
for 1 h. Samples were alkylated by addition of 200 mM
iodoacetamide (20 lL) and incubated for 1 h in the dark.
Unreacted iodoacetamide was neutralized by addition of
200 mM DTT (20 lL). The urea concentration was re-
duced by addition of dH2O (800 lL). Samples were incu-
bated with trypsin overnight at 37 ꢁC and purified on a
Vivapure C18 spin column according to the manufac-
turer’s instructions (Sartorius, Germany) (see Table 2).
Compounds yzm13 and yzm24 were obtained by coupling
the corresponding biotin or BODIPY NHS activated ester
with a 2- to 3-fold excess of compound 6 (Scheme 2) in ace-
tone withthe assistance of 2 equivofDIEA (with respect to
the amine compound). They were obtained in 90% yield
after purification by HPLC. High-resolution mass spec-
trometer (HRMS) data: [M+H]+ Calculated for yzm13,
C36H56N5O9S+, 734.38; found 734.3797. Calculated for
yzm24, C42H51BF2N5O5þ, 754.40; found 754.3955.
4.6. LC–MS/MS and database search
Samples were analyzed on a LCQ DecaXP Plus ion trap
mass spectrometer (Thermo Fisher, USA) coupled to a
nanoLC liquid chromatography unit (Eksigent, USA).
Peptides were separated on a BioBasic Picofrit C18 cap-
illary column (New Objective, USA). Elution was per-
formed with acetonitrile gradient from 0% to 50% in
the 0.1% solution of formic acid over 40 min with over-
all flowrate of 350 nL/min. The three most intense base
4.3. Preparation of mouse tissue homogenates and protein
labeling
Mouse tissue homogenates23 were incubated in sodium
acetate buffer (50 mM, pH 5.5) with yzm13 (0.1 lM final
concentration) in the presence of DTT (2 mM final con-
Table 2. MS identification of labeled proteins in liver lysate by ‘on bead’ digestion
Protein
Accession No.
MW (kDa)
Inhibitor
Probe
Cathepsin B
gi:74180941
gi:13905236
gi:22477957
gi:18655689
gi:82879179
gi:15679953
gi:26344433
gi:31982861
gi:26340966
37
80
45
16
165
33
36
29
69
3 (11%)
24 (44%)
12 (55%)
8 (60%)
7 (7%)
12 (40%)
28 (51%)
12 (43%)
8 (60%)
4 (5%)
Propionyl-coenzyme A carboxylase
Betaine–homocysteine methyltransferase
Chain D, chimeric human mouse carbonmonoxy hemoglobin
Predicted: carbamoyl-phosphate synthetase 1 isoform 1
Glycine N-methyltransferase
3 (23%)
3 (12%)
2 (18%)
2 (6%)
3 (20%)
3 (12%)
3 (18%)
3 (10%)
S-Adenosylhomocysteine hydrolase
Carbonic anhydrase 3
Albumin 1
Number of peptides identified for each protein is indicated with percentage of coverage in parentheses.

Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
1077
peaks in each scan were analyzed by MS/MS. Dynamic
exclusion was set at a repeat count of 2 with exclusion
duration of 2 min. Database searches were performed
using the mouse NCBI protein database using the Se-
quest algorithm (Thermo Fisher, USA). Peptides with
XCorr values over 1.5 (+1 charge), 2 (+2 charge) and
2.5 (+3 charge) and deltaCn values over 0.1 were consid-
ered for further evaluation. Protein and peptide hits
were statistically reevaluated by Scaffold (Proteome
Software, USA). Peptide identifications were accepted
if they could be established at greater than 95% proba-
bility as specified by the Peptide Prophet algorithm. Pro-
tein identifications were accepted if they could be
established at greater than 99.0% probability and con-
tained at least two identified peptides. Protein probabil-
ities were assigned by the Protein Prophet algorithm.23
plate one day before treatment. Cells were pre-treated
with yzm09 (10 lM), or with control DMSO (0.1%)
for 1 h and labeled for 1 h by addition of yzm24 to
culture medium. The final DMSO concentration was
maintained at 0.2%. Cells were washed with PBS
twice and lysed by addition of sample buffer (10%
glycerol, 50 mM Tris/HCl, pH 6.8, 3% SDS, and
5% b-mercaptoethanol). Lysates were boiled for
10 min and cleared by centrifugation. Equal amounts
of protein per lane were separated by 15% SDS–
PAGE, and labeled proteases were visualized by scan-
ning of the gel with a Typhoon flatbed laser scanner
(Ex/Em 532/580 nm).
Acknowledgments
4.7. Determination of kinetic rate constants of inhibition
This work was supported by the National Institutes of
Health National Technology Center for Networks and
Pathways Grant U54 RR020843, R01 EB005011, P01
CA072006, and a Stanford Chemical and Systems Biol-
ogy training grant (to S.V.).
The kinetics of inhibition was determined by the pro-
gress curve method under pseudo-first-order conditions
with at least 10-fold molar excess of inhibitor. Recorded
progress curves were analyzed by nonlinear regression
according to the following equation:
½Pꢂ ¼ mzð1 ꢀ eꢀktÞ=k
References and notes
where [P] is the product, vz is the velocity at time zero
and k is the pseudo-first-order rate constant. Apparent
rate constant (kapp) was determined from the slope of
plot k versus [I]. Because of the irreversible and compet-
itive mechanism of inhibition, kapp was converted to the
association constant (kass) using the equation below:
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kass ¼ kappð1 þ ½Sꢂ=KmÞ
Activity of human cathepsin L was measured with the
fluorogenic substrate Z-FR-AMC (Bachem; Km =
7.1 lM) and cathepsin B was assayed against the fluoro-
genic substrate Z-RR-AMC (Bachem; Km = 114 lM).
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was 10 lM. Cathepsins B and L (1 nM final concentra-
tions) were incubated with inhibitor concentrations rang-
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appropriate substrate. Total volume during the measure-
ment was 100 lL. The increase in fluorescence (370-nm
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4.9. Labeling of intact cells with probes
WT and Cat Lꢀ/ꢀ (240,000 cells per well) and Cat
Bꢀ/ꢀ (300,000 cells per well) were seeded in a six-well

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Z. Yang et al. / Bioorg. Med. Chem. 17 (2009) 1071–1078
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