Demonstration of direct binding of cIAP1 degradation-promoting bestatin analogs to BIR3 domain: Synthesis and application of fluorescent bestatin ester analogs

Article abstract of DOI:10.1016/j.bmcl.2008.04.031

Overexpression of cIAP1 correlates with resistance to radiotherapy and chemotherapy in various cancers. Recently, we reported that a class of bestatin ester analogs represented by MeBS (2) destabilized and promoted the degradation of cIAP1 through auto-ub

Full text of DOI:10.1016/j.bmcl.2008.04.031

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3354–3358
Demonstration of direct binding of cIAP1 degradation-promoting
bestatin analogs to BIR3 domain: Synthesis and application
of fluorescent bestatin ester analogs
Shinichi Sato, Hiroshi Aoyama,* Hiroyuki Miyachi,
Mikihiko Naito and Yuichi Hashimoto
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Received 10 March 2008; revised 2 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—Overexpression of cIAP1 correlates with resistance to radiotherapy and chemotherapy in various cancers. Recently, we
reported that a class of bestatin ester analogs represented by MeBS (2) destabilized and promoted the degradation of cIAP1 through
auto-ubiquitination, and thereby sensitized cancer cells to apoptosis. Herein, we present chemical evidence that bestatin ester ana-
logs directly interact with the cIAP1-BIR3 domain by means of fluorescence polarization assay and photoaffinity labeling assay
using fluorescent probes.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Programmed cell death, called apoptosis, is required for
normal embryonic development, growth, differentiation,
and homeostasis of multicellular organisms.1–4 Apopto-
sis can be triggered by distinct extracellular and intracel-
lular stimuli, and it can involve the activation of a
unique class of cysteine proteases known as caspases.5–8
The functions of caspases are regulated by another
set of proteins called inhibitor of apoptosis proteins
(IAPs).9–11
TRAF-2) in the signaling pathway mediated by tumor
necrosis factor 2 (TNFR2).16 cIAP1 and cIAP2 are
known to inhibit directly the activity of caspase-3, cas-
pase-7 and caspase-9.17,18 There are also RING do-
main-containing
ubiquitin
ligases
capable
of
promoting ubiquitination and proteasomal degradation
of several of their binding partners and themselves.17,19–22
cIAP1 is highly expressed in various organs such as
kidney, small intestine and lung, and one of the factors
causing treatment-resistance of cancer is considered to
be the apoptosis-inhibiting activity of cIAP1 in these or-
gans. Thus, the inhibition of cIAP1 function is regarded
as an attractive target for the treatment of cancer.
IAP proteins (IAPs) interact with multiple cellular part-
ners and inhibit apoptosis induced by a variety of stim-
uli.12 This places IAPs in a central position as inhibitors
of death signals that proceed through a number of differ-
ent pathways.13 The IAPs have one to three zinc-binding
baculovirus IAP repeat (BIR) domains that are required
for anti-apoptotic activity.12,14 In addition, some of
IAPs also possess carboxy-terminal RING domains that
function as ubiquitin ligases.12,15
Among human IAPs, cIAP1 and cIAP2 were originally
identified through their ability to interact directly with
TNF receptor associated factor-1 and -2 (TRAF-1,
Figure 1. FLAG-cIAP1 stable transfectant HT1080 cells were treated
with or without 3 and 30 lM bestatin (1) and MeBS (2) for 3 h. Cell
lysates were analyzed by Western blotting with the indicated
antibodies.
Keywords: Bestatin; cIAP1; BIR3 domain; Fluorescence polarization;
Photoaffinity labeling.
*
Corresponding author. Tel.: +81 03 5841 7848; fax: +81 03 5841
8495; e-mail: aoyama@iam.u-tokyo.ac.jp
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.031

S. Sato et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3354–3358
3355
MeBS (2) destabilizes cIAP1 but not cIAP2, a close
homolog of cIAP1 sharing 73% amino acid identity.
Based on this observation, we constructed a series of
chimeric molecules containing portions of cIAP1 and
cIAP2 to identify the indispensable part of cIAP1 at
which MeBS (2) elicits the degradation-promoting activ-
ity. The result of cIAP1 degradation assay using these
chimeric mutant proteins suggested a crucial role of
the BIR3 domain in MeBS-induced destabilization of
cIAP1.26 The results led us speculate that direct binding
of MeBS (2) to the BIR3 domain of cIAP1 might occur,
as has been suggested by SPR analysis.26
Figure 2. Design of bestatin ester derivatives for fluorescence polar-
ization assay and photoaffinity labeling assay.
Bestatin (1, Fig. 1), N-[(2S,3R)-3-amino-2-hydroxy-4-
phenylbutanoyl]-L-leucine, was isolated from Strepto-
myces olivoreticulithe in 1976,23 and is a potent, compet-
To develop a useful probe to examine the direct binding
of bestatin esters and cIAP1-BIR3 domain, we planned
to synthesize fluorescence-labeled bestatin esters and ap-
ply them to fluorescence polarization and photoaffinity
labeling experiments.
itive inhibitor of aminopeptidase
B and leucine
aminopeptidase.24 Bestatin (1) also possesses immuno-
modulatory effects through the stimulation of humoral
and cell-mediated immune responses and the inhibition
of aminopeptidases.25 We recently found that bestatin
alkyl ester derivatives, such as MeBS (2), possess potent
cIAP1 degradation-promoting activity as a novel bio-
logical function.26
Our previous studies showed that various derivatiza-
tions of the ester moiety of MeBS (2) could be done with
the retention of the cIAP1 degradation-promoting activ-
ity. So, we first synthesized MeBS (2) analogs with
Scheme 1. Reagents and conditions: Synthesis of fluorescence-labeled bestatin analogs. (a) Boc2O, TEA, CH2Cl2, rt, 150 min; (b) NaOH, H2O/
Acetone/MeOH (5:5:1), rt, 5 min, 91% (2 steps); (c) NaNO2, H2SO4, 0 ꢁC, 45 min, then NaN3, H2O, 4 ꢁC, 8 h, 68%; (d) PCl5, 75 ꢁC, 1 h; (e)
Ethanolamine, TEA, CH2Cl2, rt, 49% for 10a, 39% for 10b (2 steps); (f) 6, EDCI, HOBt, DIPEA, rt; (g) TFA, CH2Cl2, rt, 24% for 3 (2 steps), 13% for
4 (2 steps).

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S. Sato et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3354–3358
Figure 3. FLAG-cIAP1 stable transfectant HT1080 cells were treated
with or without 3 and 30 lM of MeBS (2), DanBE (3) and ADanBE
(4) for 3 h. Cell lysates were analyzed by Western blotting with the
indicated antibodies.
Figure 5. Competitive displacement studies were performed with
MeBS (2). To a solution of GST-BIR3 5 lM in phosphate-buffered
saline (pH 7.4), DanBE (3) was added at concentrations of 100 lM or
1 mM. After preincubation (5 min, 25 ꢁC), 2 was serially diluted in the
assay buffer to 1 lM, and the solution was incubated (5 min, 25 ꢁC).
The fluorescence polarization of 3 was measured (Ex: 335 nm, Em:
550 nm).
anthracene or BODIPY as a fluorophore, but these ana-
logs were not suitable for fluorescence polarization assay
because of their very low solubility in water. Thus, we
newly designed DanBE (3, Fig. 2) which has a dansyl
group as a fluorophore in the ester part. Moreover, we
designed ADanBE (4, Fig. 2) which has an azidodansyl
group as a fluorophore/photoaffinity labeling moiety in
the ester part.27,28
with 3 or 30 lM compound 3 or 4 for 3 h (Fig. 3). Com-
pounds 3 and 4 showed moderate cIAP1 degradation-
promoting activity. The IC50 values of MeBS (2), Dan-
BE (3), and ADanBE (4) were evaluated by a separate
set of Western blotting analysis (calculated from the de-
tected amounts of cIAP1 protein) to be 0.69, 1.86, and
18.9 lM, respectively. Though these IC50 values of com-
pounds 3 and 4 were larger than that of MeBS (2), we
expected that they act as fluorescent/photoaffinity
probes because (i) our previous studies22 indicated that
the structural requirements for cIAP1 degradation-pro-
moting activity are quite critical, and (ii) both 3 and 4
apparently possess the specific cIAP1 degradation-pro-
moting activity.
The synthesis of fluorescence-labeled bestatin esters
(DanBE; 3 and ADanBE; 4) is summarized in Scheme
1. Briefly, N-Boc-bestatin (6) was synthesized by the
protection of the amino group in MeBS (2) with a Boc
group, followed by hydrolysis at the methyl ester part
to afford 6. Azidodansyl chloride (9b) was obtained
from Laurent’s acid (7) by azidation of the amino group
and then acid chlorination of the carboxylic acid part
gave 9b. Condensation of ethanolamine and dansyl
chloride (9a) or 9b, followed by condensation with N-
Boc-bestatin (6) and alcohol (10a or 10b) afforded inter-
mediates 11a and 11b, respectively. Finally, deprotection
of the Boc group in 11a and 11b under acidic conditions
gave DanBE (3)29 and ADanBE (4),30 respectively.
Compound 3 was used as a fluorescence polarization
(FP) probe to examine its binding to cIAP1-BIR3 do-
main protein (Fig. 4). A fixed concentration of probe
(1 lM) was mixed with different concentrations of
GST-BIR3 or GST proteins (X-axis) and the FP value
(Y-axis) was measured. The FP signal rose from approx-
imately 80 mP (background mP value due to intrinsic
polarization of the probe under the experimental condi-
tions) to about 230 mP when the concentration of GST-
BIR3 was increased from 0 to 25 lM. Although the
addition of GST caused a slight increase in the FP value
(possibly because of non-specific binding), the result
clearly indicates the direct binding of compound 3 to
the BIR3 domain.
The cIAP1 degradation-promoting activity of 3 and 4
was measured by the use of FLAG-cIAP1 stably trans-
fected HT1080 cells. The transfectant cells were treated
The addition of MeBS (2) to the mixture of GST-BIR3
and compound 3 caused a dose-dependent decrease of
the FP values (Fig. 5). Although the competition seems
not to be complete, this result suggests that MeBS (2)
and compound 3 bind cIAP1-BIR3 domain in a mutu-
ally competitive manner, at least in part. The incomplete
competition and the necessity of extremely high concen-
tration of MeBS (2) to compete out DanBE (3) from
GST-BIR3 might be interpreted by (i) high non-specific
binding of 3 to GST-BIR3 and/or (ii) the nature of Dan-
BE (3) which binds GST-BIR3 with higher affinity than
MeBS (2) in spite of its lower cIAP1 degradation-pro-
moting activity than that of 2. The detailed nature of
Figure 4. Serial dilutions of GST-BIR3 or GST were incubated in the
presence of DanBE (3) (1 lM) for 5 min, 25 ꢁC in phosphate-buffered
saline (pH 7.4). After incubation, the fluorescence polarization of 3
was measured (Ex: 335 nm, Em: 550 nm).

S. Sato et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3354–3358
3357
Figure 6. GST-BIR3 and GST (100 lM) were incubated in the presence of ADanBE (4) (150 lM) for 30 min at 4 ꢁC in phosphate-buffered saline (pH
7.4). Then the mixture was irradiated with a UV lamp (365 nm) at a distance of 5 cm for 5 min at 0 ꢁC. To the reaction mixture was added phosphate-
buffered saline (pH 7.4). Non-binding 4 was removed by ultrafiltration (10000 MWCO · 3000g), and the solution was concentrated to 250 lL. The
amount of labeled GST-BIR3 or GST was measured with fluorescence counter (Ex: 345 nm, Em: 465 nm). The competitive assay was carried out
similarly, in the presence of MeBS (2) (1.5 mM).
the binding of compounds 2 and 3 to GST-BIR3 re-
mained to be investigated, but the result shown in Fig-
ure 5 afforded the evidence of the direct binding of
MeBS (2) to GST-BIR3.
Acknowledgments
We are grateful to Nippon Kayaku Co. for providing
MeBS. The work described in this letter was partially
supported by Grants-in-Aid for Scientific Research from
The Ministry of Education, Culture, Sports, Science and
Technology, Japan, and the Japan Society for the Pro-
motion of Science.
Based on the above-mentioned result, we examined the
interaction of MeBS (2) analogs with cIAP1-BIR3
domain protein using ADanBE (4) as a photoaffinity
labeling probe. GST-BIR3 and GST (negative control)
were incubated with 4 in the absence or presence of
the potent cIAP1 degradation promoter MeBS (2), and
the mixtures were irradiated with UV 365 nm. The pro-
tein fractions were separated and washed to remove
noncovalently bound ligands, and then the amount of
bound fluorescent probe to GST-BIR3 or GST was
measured with a fluorescence counter (Ex: 345 nm,
Em: 465 nm). As shown in Figure 6, GST-BIR3 was effi-
ciently labeled with compound 4 (the middle bar),
though slight non-specific labeling to GST was observed
(the left bar). The amount of covalently bound fluores-
cent probe was decreased by the addition of MeBS (2)
(the right bar, Fig. 6). Although the decrease was not
complete possibly by non-specific labeling reaction, the
results indicate that labeling occurred at the specific
MeBS (2)-binding site(s) in the BIR3 domain, at least
in part. The non-specific binding of ADanBE (4) seems
to be rather high, as well as that of DanBE (3), which
might be improved by changing the fluorescent/photore-
active moiety to more hydrophilic substitute(s). In spite
of this disadvantage of the probes, we have obtained
chemical evidence that bestatin ester analogs directly
interact with the cIAP1-BIR3 domain based on fluores-
cence polarization assay and photoaffinity labeling as-
say. Further analyses of binding of these compounds
at the cIAP1-BIR3 domain, for example, identification
of the amino acid residues involved, and the mode of
binding, are under way.
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