A bright approach to the immunoproteasome: Development of LMP2/β1i-specific imaging probes

Article abstract of DOI:10.1016/j.bmc.2011.06.039

While the constitutive, 26S proteasome plays an important role in regulating many important cellular processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive immune responses. However, recent studies indicate an association of immunoproteasomes with many physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time the development of cell-permeable activity-based fluorescent probes, UK101-Fluor and UK101-B660, which selectively target the catalytically active LMP2/β1i subunit of the immunoproteasome. These probes facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as LMP2/β1i may serve as a potential tumor biomarker, an LMP2/β1i-targeting fluorescent imaging probe may be applicable to a rapid readout assay to determine tumor LMP2/β1i levels.

Full text of DOI:10.1016/j.bmc.2011.06.039

Bioorganic & Medicinal Chemistry 20 (2012) 607–613
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A bright approach to the immunoproteasome: Development
of LMP2/b1i-specific imaging probes
Kimberly Cornish Carmony, Do-Min Lee, Ying Wu, Na-Ra Lee, Marie Wehenkel, Jason Lee, Beilei Lei,
⇑
Chang-Guo Zhan, Kyung-Bo Kim
Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
While the constitutive, 26S proteasome plays an important role in regulating many important cellular
processes, a variant form known as the immunoproteasome is thought to primarily function in adaptive
immune responses. However, recent studies indicate an association of immunoproteasomes with many
physiological disorders such as cancer, neurodegenerative, and inflammatory diseases. Despite this, the
detailed functions of the immunoproteasome remain poorly understood. Immunoproteasome-specific
probes are essential to gain insight into immunoproteasome function. Here, we describe for the first time
the development of cell-permeable activity-based fluorescent probes, UK101-Fluor and UK101-B660,
which selectively target the catalytically active LMP2/b1i subunit of the immunoproteasome. These
probes facilitate rapid detection of the cellular localization of catalytically active immunoproteasomes
in living cells, providing a valuable tool to analyze immunoproteasome functions. Additionally, as
LMP2/b1i may serve as a potential tumor biomarker, an LMP2/b1i-targeting fluorescent imaging probe
may be applicable to a rapid readout assay to determine tumor LMP2/b1i levels.
Received 4 April 2011
Revised 31 May 2011
Accepted 14 June 2011
Available online 7 July 2011
Keywords:
Immunoproteasome
Fluorescent probes
Near-infrared fluorescent probe
LMP2
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
The important role of the ubiquitin–proteasome system (UPS)
in many essential cellular processes is now well-documented.1,2
The proteasome, a key component of the UPS, is a multi-protease
complex responsible for the degradation of poly-ubiquitinated pro-
teins involved in many important biological processes such as the
cell cycle, development, and inflammatory responses. The immu-
noproteasome is a variant form of the constitutive proteasome.
Normally, the immunoproteasome is constitutively expressed in
cells of hematopoietic origin but can also be induced in other cell
types by cytokines such as interferon-
c (IFN-c) or tumor necrosis
factor- (TNF-
a
a
).3 Upon exposure to these stimuli, the immuno-
proteasome catalytic subunits LMP2/b1i, MECL1/b2i, and LMP7/
b5i, are synthesized and incorporated, replacing their constitutive
proteasome counterparts Y/b1, Z/b2 and X/b5, respectively
(Fig. 1). Similar to those of the constitutive proteasome, catalytic
subunits of the immunoproteasome are synthesized as inactive
forms containing N-terminal propeptides: pre-LMP2, pre-LMP7,
and pre-MECL1. Upon completion of proteasome assembly, the
N-terminal propeptides of these inactive catalytic subunits are re-
moved to expose the catalytic threonine residues.4,5 In comparison
with the constitutive proteasome, the capacity of the immunopro-
Figure 1. Schematic representation of immunoproteasome and constitutive pro-
teasome assembly.
teasome to generate peptides bearing C-terminal hydrophobic
amino acids is enhanced, while its capacity to produce peptides
bearing C-terminal acidic residues is reduced.6 This results in the
increased production of peptides which can associate with MHC
class I molecules for antigen presentation.
Although the immunoproteasome is thought to primarily func-
tion in adaptive immune responses, several recent studies have
shown that its cellular roles are not limited to the generation of
⇑
Corresponding author.
E-mail address: kbkim2@uky.edu (K.-B. Kim).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.039

608
K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
used for real-time monitoring of proteasome activity for correla-
tion with cellular processes. One example of a synthetic protea-
some-targeting fluorescent probe is BODIPY–epoxomicin, which
binds selectively to the b5/b1 proteasome subunits (Fig. 2).27 An-
other example is MV151,26 which was prepared by a medicinal
chemistry approach and binds to all catalytic subunits of the pro-
teasome in living cells. This kind of fluorescent probe can be used
for the clinical profiling of proteasome activity, biochemical analy-
sis of the subunit specificity of inhibitors, or cell biological analysis
of proteasome functions and dynamics. Furthermore, Berkers et al.
have shown that these fluorescently labeled ABPs can be used to
study functionally active proteasomes in tissues.28 Despite the
development of several fluorescently labeled ABPs targeting pro-
teasomes,28,29 there are currently no available immunoprotea-
some- or constitutive proteasome-specific ABPs that can be used
to dissect the distinctive functions of proteasome subtypes.
In this article, we report the synthesis and characterization of
fluorescent ABPs that selectively target the immunoproteasome
in living cells. These probes enable fast labeling of catalytically ac-
tive immunoproteasomes and can therefore be used to visualize
their cellular localization. This information may provide an impor-
tant clue in understanding immunoproteasome functions in living
cells. Furthermore, given that LMP2/b1i is highly expressed in sev-
eral types of cancer, such an LMP2-targeting ABP may potentially
serve as a tool for cancer screening as well as for monitoring cancer
progression.
Figure 2. Structures of BODIPY-labeled epoxomicin and MV151. BODIPY-labeled
epoxomicin selectively targets constitutive proteasome subunits b1, b5 and, to a
lesser extent, LMP7, while MV151 covalently modifies all catalytically active
proteasome subunits.
antigenic peptides but are far more multifaceted.7–9 For example,
the immunoproteasome has been implicated in a number of path-
ological disorders such as cancer, neurodegenerative and autoim-
mune diseases.10–15 More recently, the immunoproteasome has
been shown to clear protein aggregates that accumulate under oxi-
dative stress.8 Despite these contributions, our understanding of
immunoproteasome function still remains limited.
2. Results and discussion
While recent advances in proteomics allow global proteome
profiling, mechanistic dissection of the correlation between the
proteome information and protein dynamics in the microcellular
environment or disease states is still a major challenge in biology.
One of the primary goals of chemical biology is to investigate such
a biological conundrum using small molecules that perturb signal-
ing proteins or pathways. Use of these small molecules has proven
to be an effective strategy to decipher protein functions within
cells.16 Pioneered by Cravatt and Bogyo, the use of small molecules
has further evolved to exploit their function as active site-directed
activity-based probes (ABPs), allowing the monitoring, in real time,
of the activities of proteases in disease states.17–24 Typically, an
ABP targets an enzyme active site to monitor the functional en-
zyme in living cells. General proteasome inhibitors have also been
exploited as ABP imaging agents that report proteasome activities
in cells. Fluorescently labeled proteasome inhibitors25,26 can be
2.1. Synthesis of fluorescein-labeled UK-101
Given that UK-101 (Fig. 3), a small molecule inhibitor previously
developed in our laboratory,10 selectively targets LMP2 in cells, we
wanted to use UK-101 as a lead compound in developing LMP2-tar-
geting fluorescent probes. Our molecular modeling studies30 indi-
cated that the alanine side chain at the P2 site of UK-101 is
exposed towards the surface of LMP2 through the open channel
extending from the active site (Fig. 3A). This suggests that the ala-
nine side chain of UK-101 can be replaced by other linear groups
without disrupting the specific UK-101:LMP2 interaction (Fig. 3B).
Therefore, we set out to introduce a linear hydrocarbon linker at
the P2 position having a free amine functional group, which can
be used as a handle to attach a fluorophore to UK-101 (Fig. 3B).
Figure 3. Proposed LMP2 binding mode and derivatization of UK-101. A) Molecular model depicting the binding of UK-101 (stick model) within the catalytic cleft of LMP2
(surface model). B) Structure of UK-101. The alanine side chain at the P2 position is indicated by the blue circle. This side chain was derivatized for the attachment of the
fluorescein or BODIPY fluorophore to give UK101-Fluor or UK101-B660.

K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
609
Scheme 1. Synthesis of UK101-Fluor (9) and UK101-B660 (10). Fluorescein or BODIPY 650/665 (Invitrogen) was attached at the P2 position of UK101-Lys (8). The first
coupling between compounds 5 and 6 was performed using standard peptide coupling agents: HBTU (1 equiv), HOBt (1 equiv), DIPEA (5 equiv) and CH2Cl2 (reaction solvent).
Specifically, the key intermediate, UK101-Lys-Cbz (7), was first pre-
pared following a procedure similar to that reported for the synthe-
sis of UK-101,10 replacing alanine with Cbz-protected lysine
(Scheme 1). Before coupling a fluorescent residue to UK-101 via
the lysine linker, we wanted to ensure that this intermediate (7)
maintained binding selectivity for LMP2. This was first confirmed
in EL4 mouse lymphoma cells, which highly express both the
immunoproteasome and the constitutive proteasome. The cells
were treated with UK101-Lys-Cbz (7) or DMSO (vehicle control),
lysed, and subjected to western blotting. As shown by an increase
in molecular weight resulting from covalent modification,
UK101-Lys-Cbz binds to LMP2 (Fig. 4A). To identify the minimum
concentration of UK101-Lys-Cbz required for complete covalent
modification of LMP2, EL4 cells were treated with a range of low
micromolar concentrations of UK101-Lys-Cbz, DMSO, or the
general proteasome inhibitor epoxomicin. The cells were then lysed
and subjected to western blotting as before. The results revealed
that 1
a 5 M concentration of this compound labels most of the LMP2 in
these cells, as visualized by a partially incomplete upward shift of
lM of UK101-Lys-Cbz is not sufficient to modify LMP2, while
l
Figure 4. Binding specificity of the UK101-Lys-Cbz (8) and UK-101 (3). EL4 (A and C) or PC-3 (B, D, and E) cells were treated with DMSO (vehicle control) or increasing
concentrations of UK101-Lys-Cbz or UK-101, lysed, and subjected to western blotting. Subunit binding specificity was determined using antibodies recognizing proteasome
and immunoproteasome subunits. An upward shift of a band with respect to the DMSO control indicates an increase in molecular weight due to covalent modification. 1
lM
epoxomicin (Epx)-treated cells served as a positive control for LMP2, LMP7, and X band shifts in (C), and for LMP7 and X band shifts in (D and E). However, epoxomicin does
not bind Y under these conditions, and therefore no shift is observed for this subunit in (C). For (D and E), 10 lM YU-102-treated cells served as a positive control for LMP2
and Y band shifts.

610
K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
the LMP2 band (Fig. 4C). A 10
in complete covalent modification of LMP2, displayed by a com-
plete upward shift of the LMP2 band (Fig. 4C). In contrast, this inter-
l
M dose of UK101-Lys-Cbz resulted
cent probe, and visualized via fluorescence microscopy. Binding of
UK101-Fluor was observed to be dose-dependent (Fig. 5A). Based
on this result, we selected a concentration of 15 lM for further
mediate (7) does not bind LMP7, Y, or X at 10
l
M (Fig. 4C). Similar
studies, as this concentration gave the optimum signal to back-
ground ratio. We then wanted to ensure that the fluorescent signal
observed resulted from UK101-Fluor binding to LMP2. To do this,
we performed a competition assay in living cells. Specifically, PC-3
cells were preincubated with UK-101 (3) under conditions that
result in complete covalent modification of LMP2, but not other pro-
teasome subunits, as detected by a mobility shift assay (Fig. 4E).
Following this preincubation, the cells were treated with UK101-
Fluor, washed to remove unbound probe, and visualized via fluores-
cence microscopy. Pretreatment with UK-101 effectively competed
away the signal observed upon UK101-Fluor treatment alone, sug-
gesting that UK101-Fluor selectively targets LMP2 (Fig. 5B). To con-
firm this result, PC-3 cells stably transfected with the empty pLKO
vector or with the pLKO vector containing a short hairpin RNA
against LMP2 were treated with UK101-Fluor as in Figure 5A. The
signal observed from UK101-Fluor in cells transfected with the
emptyvector was markedly decreased in the LMP2 knockdown cells,
further indicating the selectivity of UK101-Fluor for LMP2 (Fig. 5C).
results were obtained using the PC-3 human prostate cancer cell
line (Fig. 4B and D). It should be noted that synthesis of proteasome
subunits can be induced in response to treatment with proteasome
inhibitors, explaining the variation in band intensity.31 Collectively,
these results suggest that derivatization of UK-101 via a linear
hydrocarbon chain at the P2 position does not influence the
LMP2-binding specificity of UK-101. Encouraged by this, UK101-
Lys-Cbz (7) was hydrogenated and coupled to fluorescein using
fluorescein-NHS (fluorescein N-succinimidyl ester) to yield
UK101-Fluor (9) (Scheme 1).
2.2. Visualization of catalytically active LMP2 using UK101-
Fluor
Next, we investigated whether UK101-Fluor (9) facilitates visual-
ization of LMP2 in living cells. First, PC-3 cells were treated with
UK101-Fluor (9), washed extensively to remove unbound fluores-
Figure 5. UK101-Fluor (9) selectively binds to LMP2 in PC-3 cells. A) UK101-Fluor binding with LMP2 is dose-dependent. PC-3 cells were treated with increasing
concentrations of UK101-Fluor. After extensive washing to remove unbound probe, binding was examined via fluorescence microscopy. Fluorescence intensity of all images
was normalized to DMSO control. Of note, LMP2 binding was also observed in cells treated with 5 lM UK101-Fluor with a longer exposure time. B) Pre incubation with UK-
101 competes away the signal observed following treatment with UK101-Fluor alone. PC-3 cells pretreated with UK-101 or DMSO were subsequently treated with UK101-
Fluor, fixed, and examined via fluorescence microscopy. Fluorescence intensity in all images was normalized to control (-UK-101/-UK101-Fluor). C) LMP2 knockdown results
in decreased binding of UK101-Fluor. PC-3 cells transfected with either the empty pLKO vector (LMP2 Knockdown -) or the pLKO vector containing a short hairpin RNA
against LMP2 (LMP2 knockdown +) were treated with DMSO or UK101-Fluor, fixed, and examined via fluorescence microscopy. Fluorescence intensity in all images was
normalized to controls (UK101-Fluor -). D) UK101-Fluor cellular localization. Cells were incubated with UK101-Fluor and subsequently stained with antibodies recognizing
LMP2 or calnexin, an endoplasmic reticulum (ER) marker. The cells were then fixed and examined via fluorescence microscopy. The arrows in the merged images point to
areas of overlap between green and red staining, which gives a yellow color.

K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
611
To examine the cellular localization pattern of catalytically ac-
tive LMP2, cells were treated with UK101-Fluor (9) as before, but
were then fixed and stained with an antibody detecting LMP2. It
should be noted that UK101-Fluor (9) exclusively detects catalyti-
cally active LMP2, which only exists within fully assembled protea-
somes, in contrast to the LMP2 antibody, which detects both
catalytically active as well as inactive or pre-LMP2. As shown in
Figure 5D (top panel), catalytically active LMP2 was easily detected
by UK101-Fluor and does not display the diffuse distribution ob-
served of the total cellular population of LMP2. In contrast, the
total population of LMP2 appears to be diffusely distributed
throughout the cell (Fig. 5D). To investigate whether catalytically
active LMP2 is localized near the endoplasmic reticulum (ER), we
stained fixed UK101-Fluor-treated cells with an antibody recogniz-
ing calnexin, an ER marker. As shown in Figure 5D (bottom panel),
the majority of catalytically active LMP2 appears to be localized to
the ER, as reported.32,33 Taken together, these results demonstrate
that the LMP2-specific inhibitor UK-101 can be derivatized for the
development of immunoproteasome imaging probes, providing
powerful immunoproteasome imaging tools.
LMP2 may provide a novel diagnostic tool. Given the successful
development of UK101-Fluor (Fig. 5), we next sought to develop
a near-infrared fluorescent (NIRF) probe that selectively targets
LMP2 and can be applied to animal model studies. Specifically,
UK101-Lys-Cbz (7) was hydrogenated and coupled to a near-infra-
red fluorescent group (BODIPY 650/665) using BODIPY 650/665-SE
to yield UK101-B660 (10) (Scheme 1).
2.4. Visualization of catalytically active LMP2 using UK101-B660
To verify that UK101-B660 (10) selectively targets LMP2, we first
performed an in-gel fluorescence assay using a Typhoon imaging
system. Extracts from cells treated with UK101-B660 (10) were
subjected to gel electrophoresis, and the gel was imaged to deter-
mine the molecular weight of proteins covalently modified by this
compound. As shown in Figure 6A, a protein band with a size of
ꢀ23 kDa appeared, suggesting that the UK101-B660-labeled protein
is likely an immunoproteasome subunit. To further confirm that this
labeled protein is LMP2, a competition assay was performed with
UK-101 (3) and UK101-Lys-Cbz (7). Specifically, cells were
pretreated with UK-101 (3) or UK101-Lys-Cbz (7), followed by treat-
ment with UK101-B660 (10). Cell lysates were then prepared and
analyzed as in Figure 6A. As shown in Figure 6B, preincubation with
UK-101 or UK101-Lys-Cbz significantly attenuated the fluorescent
signal resulting from UK101-B660 treatment, indicating that
2.3. Synthesis of a near-infrared fluorescent probe
LMP2 is highly expressed in various types of cancer,10,14,15,34,35
and therefore a non-invasive imaging probe that selectively detects
Figure 6. UK101-B660 (10) selectively binds to LMP2 in PC-3 cells. A and B) UK101-B660 targets the immunoproteasome catalytic subunit LMP2. A) UK101-B660 binding is
dose-dependent. PC-3 cells were treated with DMSO (vehicle control), the general proteasome inhibitor epoxomicin (Epx), or increasing concentrations of UK101-B660. The
cells were then lysed, and proteins were resolved by SDS-PAGE. Gels were imaged using a Typhoon imager. M = Marker. B) UK-101 or UK101-Lys preincubation competes
away the fluorescent signal of UK101-B660. PC-3 cells were preincubated with DMSO (vehicle control), UK-101, or UK101-Lys, followed by incubation with DMSO or UK101-
B660. The cells were then lysed, and the proteins were resolved and imaged as in (A). M = Marker. C) Cells treated with UK-101 display a red fluorescent signal that is
competed away by UK-101 pretreatment. PC-3 cells were pretreated with either DMSO (vehicle control) or UK-101 and subsequently treated with DMSO or UK101-B660.
UK101-B660 binding was then visualized via fluorescence microscopy.

612
K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
UK101-B660 specifically binds LMP2 rather than another subunit.
To further verify this, we performed a similar competition assay
using a fluorescence microscopy readout. As shown in Figure 6C,
pretreatment of cells with UK-101 or UK101-Lys-Cbz effectively
depleted fluorescent signals derived from UK101-B660, consistent
with the results obtained from the in-gel fluorescence assay. Addi-
tionally, the cellular localization of UK101-B660 appeared to take
on a similar pattern as that of UK101-Fluor (compare Fig. 5B and D
with Fig. 6C). To ensure that LMP2 was still present and maintained
NP-40, 1% Triton X-100, and 1% protease inhibitor cocktail (Sig-
ma–Aldrich). The lysates were then centrifuged at 14,000 rpm at
4 °C for 10 min (Sorvall Biofuge Primo R, Kendro Laboratory Prod-
ucts) and the supernatants were collected. Protein concentrations
of the supernatants were determined by the Bradford method
using Protein Assay Dye Reagent Concentrate (Bio-Rad) and a
GENESYS 10 spectrophotometer, Thermo Spectronic (VWR). 2X
Laemmli Sample Buffer (Sigma–Aldrich) was added to the superna-
tants, which were then heated on a heat block at 100 °C for 10 min.
Equal protein concentrations of the resulting samples were then
loaded onto 12% polyacrylamide gels (Bio-Rad), resolved by SDS–
PAGE, and transferred onto PVDF membranes (Bio-Rad). Mem-
branes were blocked on a rotating platform for 1 h at room temper-
ature in 5% nonfat dry milk (Bio-Rad). Primary and secondary
antibodies were diluted in 3% BSA and incubated with membranes
for 1 h at room temperature on a rotating platform or overnight at
4 °C. Proteins were visualized on Kodak BioMax XAR Films (Sigma–
Aldrich) using ECL Western Blotting Substrate (Pierce).
a
normal cellular distribution upon UK-101 treatment, cells
pretreated with UK-101 followed by treatment with UK101-B660
were also stained with an antibody recognizing LMP2. As shown in
Figure 6C (bottom panel), UK-101 treatment did not impact the lev-
els or cellular distribution of LMP2 (compare with Fig. 5D, top panel).
Together, these results suggest that UK101-B660 can be used as an
imaging probe to visualize catalytically active LMP2 in living cells.
3. Conclusions
4.4. In-gel fluorescence
In summary, for the first time, we have successfully developed
fluorescent imaging agents that specifically target the catalytically
active immunoproteasome subunit LMP2. Given that the immuno-
proteasome is gaining increasing attention as a therapeutic target
for cancer, these fluorescent probes will provide a powerful tool
to investigate therapeutic potentials of targeting the immunopro-
teasome as well as functions of the immunoproteasome yet to be
discovered. Additionally, with LMP2 serving as a potential tumor
biomarker, our NIRF LMP2-targeting probe may be directly appli-
cable to in vivo screening in animal models.
Denatured whole cell lysates were prepared and proteins were
resolved as described for immunoblotting. Gels were then imaged
using
a Typhoon FLA 9000 imaging system (kex = 635 nm,
kem = 665).
4.5. Fluorescence microscopy
Cells were plated on coverslips or chamber slides and incubated
for 24–48 h prior to treatment. Following treatment, the cells were
fixed with 4% paraformaldehyde in PBS for 7 min at 37 °C and per-
meablized with 0.2% Triton-X in PBS for 30 min at 37 °C. Coverslips
or slides were then blocked in 10% goat serum and 1% BSA in PBST.
Where indicated, anti-LMP2 or anti-calnexin primary antibodies
were diluted in DakoCytomation Antibody Diluent with Back-
ground Reducing Components and incubated with cells for 1 h at
37 °C. Secondary antibodies were similarly diluted and incubated
with cells for 30 min at 37 °C. Following this incubation, coverslips
or slides were extensively washed with PBST to remove unbound
ABP or antibody. Coverslips were mounted onto microscope slides
using Prolong Gold antifade reagent with DAPI (Invitrogen) or VEC-
TASHIELD Mounting Medium with DAPI (Vector Laboratories).
Cells were then visualized using an inverted Nikon-Ti-U fluores-
cence microscope and NIS Element Research image analysis
software.
4. Experimental
4.1. Cell culture
EL4 and PC-3 cell lines were obtained from the American Type
Culture Collection. These cells were cultured according to the man-
ufacturer’s instructions at 37 °C in 5% CO2. PC-3 cells stably trans-
fected with the empty pLKO vector or the pLKO vector containing a
short hairpin RNA against LMP2 were maintained in F-12 K med-
ium containing 0.5 lg/mL puromycin at 37 °C in 5% CO2.
4.2. Compounds
UK-101 and epoxomicin were synthesized as previously de-
scribed.10,36 Fluorescent probes (9 and 10) were synthesized fol-
lowing a procedure similar to that previously reported.10
4.6. UK101-Lys-Cbz treatment
Compound 9, 1H NMR (500 MHz, CDCl3): d 8.05 (s, 1H), 7.94 (s,
1H), 6.94–6.88 (m, 1H,), 6.63–6.56 (m, 3H), 4.83 (m, 8H), 4.49–4.46
(m, 1H), 4.33–4.27 (m, 1H), 3.51–3.27 (m, 3H), 2.96 (s, 4H), 2.82 (s,
4H), 2.61 (s, 3H), 2.20–2.14 (m, 2H), 1.79–1.42 (m, 9H), 1.40–1.11
(m, 11H), 0.90–0.84 (m, 18H), 0.06–0.01 (m, 6H); MS-spec: calcd
900.45 (M+H)+, found 900.45 (M+H)+.
EL4 or PC-3 cells were treated with DMSO, 1 lM epoxomicin, or
UK101-Lys-Cbz at the indicated concentrations for 1.5 h. The cells
were then washed with Hank’s Balanced Salt Solution (Gibco) and
harvested for immunoblotting.
Compound 10, 1H NMR (500 MHz, CDCl3): d = 7.592 (d, 2H,
J = 13.5 Hz), 7.230 (d, 2H, J = 13.5 Hz), 7.058 (s, 1H), 6.974 (m,
2H), 6.927 (m, 1H), 6.889 (m, 2H), 6.823 (m, 1H), 6.643(s, 1H),
6.399(s, 1H), 5.387 (s, 1H), 5.351(m, 1H), 4.556 (m, 3H), 4.429
(dd, 2H, J = 16.0, 16.0 Hz), 3.659 (m, 4H), 3.519 (m, 1H), 2.999 (m,
1H), 2.103–2.230 (m, 4H), 2.011–2.056 (m, 4H), 1.459–1.645 (m,
7H), 1.080–1.436 (m, 12H), 0.851–1.029 (m, 18H), 0.046 (s, 6H).
MS-spec: calcd 1069.61 (M+H)+, found 1069.7 (M+H)+.
4.7. UK101-Fluor and UK101-B660 treatment
PC-3 cells were treated with DMSO, UK101-Fluor or UK101-
B660 at the indicated concentrations for 4 h. The cells were then
washed with Hank’s Balanced Salt Solution (Gibco) and harvested
for an in-gel fluorescence assay or prepared for fluorescence
microscopy.
4.8. Competition assays
4.3. Immunoblotting
For the in-gel fluorescence competition assay, PC-3 cells were
To prepare whole cell lysates, cells were incubated for 1 h on ice
in non-denaturing lysis buffer (50 nM Tris–Cl, 150 mM NaCl, 1%
pretreated with DMSO, 1
lM UK-101 or 20 lM UK101-Lys for
1.5 h. 30 M of UK101-B660 was then added to the cell culture
l

K. C. Carmony et al. / Bioorg. Med. Chem. 20 (2012) 607–613
613
14. Altun, M.; Galardy, P. J.; Shringarpure, R.; Hideshima, T.; LeBlanc, R.; Anderson,
K. C.; Ploegh, H. L.; Kessler, B. M. Cancer Res. 2005, 65, 7896.
15. Mishto, M.; Bellavista, E.; Santoro, A.; Stolzing, A.; Ligorio, C.; Nacmias, B.;
Spazzafumo, L.; Chiappelli, M.; Licastro, F.; Sorbi, S.; Pession, A.; Ohm, T.;
Grune, T.; Franceschi, C. Neurobiol. Aging 2006, 27, 54.
16. Ovaa, H.; van Leeuwen, F. ChemBioChem 2008, 9, 2913.
17. Nomura, D. K.; Dix, M. M.; Cravatt, B. F. Nat. Rev. Cancer 2010, 10, 630.
18. Fonovic, M.; Verhelst, S. H.; Sorum, M. T.; Bogyo, M. Mol. Cell. Proteomics 2007.
19. Sadaghiani, A. M.; Verhelst, S. H.; Bogyo, M. Curr. Opin. Chem. Biol. 2007, 11, 20.
20. Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko, C.; Rice, M. J.; Sloane,
B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 203.
medium, and the cells were incubated for an additional 4 h. For
fluorescence microscopy-based competition assays, PC-3 cells
were pretreated with DMSO or 5 lM UK-101for 1.5 h. 15 lM of
UK101-Fluor or UK101-B660 was then added to the cell culture
medium, and the cells were incubated for an additional 4 h. The
cells were then harvested for an in-gel fluorescence assay or pre-
pared for fluorescence microscopy.
Acknowledgements
21. Blum, G.; von Degenfeld, G.; Merchant, M. J.; Blau, H. M.; Bogyo, M. Nat. Chem.
Biol. 2007, 3, 668.
22. Berger, A. B.; Vitorino, P. M.; Bogyo, M. Am. J. Pharmacogenomics 2004, 4, 371.
23. Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Annu. Rev. Biochem. 2008, 77, 383.
24. Kikuchi, K. Chem. Soc. Rev. 2010, 39, 2048.
25. Verdoes, M.; Berkers, C. R.; Florea, B. I.; van Swieten, P. F.; Overkleeft, H. S.;
Ovaa, H. Methods Mol. Biol. 2006, 328, 51.
This work has been supported by the Markey Cancer Founda-
tion and NIH (CA131059). We thank members of the Kim lab for
their helpful comments on the manuscript.
26. Verdoes, M.; Florea, B. I.; Menendez-Benito, V.; Maynard, C. J.; Witte, M. D.; van
der Linden, W. A.; van den Nieuwendijk, A. M.; Hofmann, T.; Berkers, C. R.; van
Leeuwen, F. W.; Groothuis, T. A.; Leeuwenburgh, M. A.; Ovaa, H.; Neefjes, J. J.;
Filippov, D. V.; van der Marel, G. A.; Dantuma, N. P.; Overkleeft, H. S. Chem. Biol.
2006, 13, 1217.
27. Verdoes, M.; Hillaert, U.; Florea, B. I.; Sae-Heng, M.; Risseeuw, M. D.; Filippov,
D. V.; van der Marel, G. A.; Overkleeft, H. S. Bioorg. Med. Chem. Lett. 2007, 17,
6169.
28. Berkers, C. R.; van Leeuwen, F. W.; Groothuis, T. A.; Peperzak, V.; van Tilburg, E.
W.; Borst, J.; Neefjes, J. J.; Ovaa, H. Mol. Pharm. 2007.
29. Berkers, C. R.; Verdoes, M.; Lichtman, E.; Fiebiger, E.; Kessler, B. M.; Anderson,
K. C.; Ploegh, H. L.; Ovaa, H.; Galardy, P. J. Nat. Methods 2005, 2, 357.
30. Lei, B.; Abdul Hameed, M. D.; Hamza, A.; Wehenkel, M.; Muzyka, J. L.; Yao, X. J.;
Kim, K. B.; Zhan, C. G. J. Phys. Chem. B 2010.
References and notes
1. Petroski, M. D. BMC Biochem. 2008, 9(Suppl 1), S7.
2. Marques, A. J.; Palanimurugan, R.; Matias, A. C.; Ramos, P. C.; Dohmen, R. J.
Chem. Rev. 2009, 109, 1509.
3. Kloetzel, P. M. Nat. Rev. Mol. Cell Biol. 2001, 2, 179.
4. Griffin, T. A.; Nandi, D.; Cruz, M.; Fehling, H. J.; Kaer, L. V.; Monaco, J. J.; Colbert,
R. A. J. Exp. Med. 1998, 187, 97.
5. De, M.; Jayarapu, K.; Elenich, L.; Monaco, J. J.; Colbert, R. A.; Griffin, T. A. J. Biol.
Chem. 2003, 278, 6153.
6. Rivett, A. J.; Hearn, A. R. Curr. Protein Pept. Sci. 2004, 5, 153.
7. Groettrup, M.; Kirk, C. J.; Basler, M. Nat. Rev. Immunol. 2010, 10, 73.
8. Seifert, U.; Bialy, L. P.; Ebstein, F.; Bech-Otschir, D.; Voigt, A.; Schroter, F.;
Prozorovski, T.; Lange, N.; Steffen, J.; Rieger, M.; Kuckelkorn, U.; Aktas, O.;
Kloetzel, P. M.; Kruger, E. Cell 2010, 142, 613.
31. Meiners, S.; Heyken, D.; Weller, A.; Ludwig, A.; Stangl, K.; Kloetzel, P. M.;
Kruger, E. J. Biol. Chem. 2003, 278, 21517.
9. Yewdell, J. W. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 9089.
10. Ho, Y. K.; Bargagna-Mohan, P.; Mohan, R.; Kim, K. B. Chem. Biol. 2007, 14, 419.
11. Kuhn, D. J.; Hunsucker, S. A.; Chen, Q.; Voorhees, P. M.; Orlowski, M.; Orlowski,
R. Z. Blood 2009, 113, 4667.
12. Fitzpatrick, L. R.; Khare, V.; Small, J. S.; Koltun, W. A. Dig. Dis. Sci. 2006, 51, 1269.
13. Fitzpatrick, L. R.; Small, J. S.; Poritz, L. S.; McKenna, K. J.; Koltun, W. A. Dis. Colon
Rectum 2007, 50, 337.
32. Rivett, A. J. Curr. Opin. Immunol. 1998, 10, 110.
33. Brooks, P.; Murray, R. Z.; Mason, G. G.; Hendil, K. B.; Rivett, A. J. Biochem. J.
2000, 352(Pt 3), 611.
34. Huang, Y. C.; Chuang, L. Y.; Hung, W. C. Mol. Pharmacol. 2002, 62, 1515.
35. Mishto, M.; Santoro, A.; Bellavista, E.; Bonafe, M.; Monti, D.; Franceschi, C.
Ageing Res. Rev. 2003, 2, 419.
36. Ho, A.; Cyrus, K.; Kim, K. B. Eur. J. Org. Chem. 2005, 4829.