Chemical affinity matrix-based identification of prohibitin as a binding protein to anti-resorptive sulfonyl amidine compounds

Article abstract of DOI:10.1016/j.bmcl.2010.11.123

In order to identify the binding proteins to anti-resorptive 5-chloro-1-(2,6-dimethylpiperidin-1-yl)-N-tosylpentan-1-imine (1), the chemical affinity matrix for the compound 1 (2b) was designed and synthesized. Using 2b-based chemical proteomics, prohibit

Full text of DOI:10.1016/j.bmcl.2010.11.123

Bioorganic & Medicinal Chemistry Letters 21 (2011) 727–729
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical affinity matrix-based identification of prohibitin as a binding protein
to anti-resorptive sulfonyl amidine compounds
Sung-Youn Chang a, , Su Jung Bae b, , Myung Yun Lee a, Seung-hwa Baek b, Sukbok Chang c,
Seong Hwan Kim b,
⇑
a Medicinal Chemistry Research Center, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea
b Laboratory of Chemical Genomics, Pharmacology Research Center, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong-gu, Daejeon 305-600, Republic of Korea
c Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST), Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to identify the binding proteins to anti-resorptive 5-chloro-1-(2,6-dimethylpiperidin-1-yl)-N-
tosylpentan-1-imine (1), the chemical affinity matrix for the compound 1 (2b) was designed and synthe-
sized. Using 2b-based chemical proteomics, prohibitin was identified as one of strong binding proteins
for 2b.
Received 20 October 2010
Revised 25 November 2010
Accepted 30 November 2010
Available online 10 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Chemical affinity matrix
Chemical proteomics
Prohibitin
The identification of target or binding proteins for bioactive
small molecules including new therapeutics can extend our under-
standing of their action mechanism (or responses) and provide the
biochemical evidence for the prediction of their side effects in the
body. Furthermore, the discovery of new drugable target protein(s)
through the process of target identification may offer an improved
therapeutic benefit by accelerating the development of new thera-
peutics based on them. Therefore, the chemical proteomics with a
fundamental goal to identify target (or binding) proteins for bioac-
tive small molecules is an important field in the view of drug
discovery and development as well as the basic research although
it is required for the advanced, complicated and integrated tools
and technologies for the purification, enrichment, identification
and validation of target proteins.1,2
Osteoclastogenesis is the overall process of hematopoietic stem
cells to differentiate into mature osteoclasts that resorb the bone
matrix, but the resorptive area is filled with bone matrix synthe-
sized by osteoblasts.3,4 This is referred as bone remodeling. How-
ever, an imbalance in bone remodeling caused by increased bone
resorption over bone formation due to several factors such as
menopause leads to the decrease of bone mineral density and it
subsequently increases the risk of factures that is a major cause
of mortality in the patients with bone disorders such as
osteoporosis.
Previously, we reported that sulfonyl amidine derivatives
exhibited anti-resorptive activities to inhibit the osteoclastogene-
sis.5 Among the sulfonyl amidine derivatives, 5-chloro-1-(2,6-dim-
ethylpiperidin-1-yl)-N-tosylpentan-1-imine (1) was proved as a
potent anti-resorptive agent with the IC50 value of 1.75 0.26
(Fig. 1).
lM
To identify the target protein of the amidine compound 1, a
chemical affinity matrix 2b was designed (Fig. 1). Several ap-
proaches have been developed to purify/enrich and identify target
(or binding) proteins/peptides.1 Among them, the photoaffinity
probe can strongly and irreversibly bind to its target proteins,
Cl
N
S
R
N
O
O
2
N
O
S
N
O
O
1
2
2a R = NHBoc-
2b R =
O
H
H
N
N
O
N
H
agarose
⇑
Corresponding author. Tel.: +82 42 860 7687.
E-mail address: hwan@krict.re.kr (S.H. Kim).
O
O
S.Y.C. and S.J.B. equally contributed to this study.
Figure 1. Structure of chemicals.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.123

728
S.-Y. Chang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 727–729
but it could also crosslink with the non-specific proteins. Addition-
ally, the introduction of a photoreactive moiety and a tag such as
biotin into the structure of bioactive small molecule causes the de-
crease of biological activity and any problem due to the condition
to separate the protein–probe covalent complex in the affinity col-
umn (e.g. normally, breaking the avidin–biotin interaction requires
6–8 M guanidine–HCl, pH 1.5) during analysis by MALDI should be
avoided by dilution or washing of the protein sample. Therefore, in
this study, the chemical affinity matrix-based approach was simply
introduced as the tool to fish out the binding partners. Generally,
the biological activity of small molecule can be decreased by
introducing a linker for target protein identification. So the appro-
priate decision of linker’s size and the site for its attachment are
critical for the design of the chemical affinity matrix. When the
2,6-dimethylpiperidine moiety of amidine 1 was replaced with
diisopropylamino group, the resulting amidine compound showed
no anti-resorptive activity (105% TRAP activity of control at 60 lM)
implying that the 2,6-dimethylpiperidine moiety is necessary for
its anti-resorptive activity. TRAP activity was evaluated as de-
scribed previously.5 Therefore, the 2,6-dimethylpiperidine moiety
was retained for the chemical affinity matrix. Next, the 4-chlorobu-
tyl moiety was changed into tert-butyl 2-(2-methoxyethoxy)eth-
ylcarbamate group, which can generate the amine group for the
coupling with the Affi-Gel, and the resulting amidine 2a showed
comparable inhibitory effect on TRAP activity with the IC50 value
of 6.89 0.69 lM to the compound 1 (Fig. 1).
Therefore, the chemical affinity matrix (2b; Fig. 1) containing
2-(2-methoxyethoxy)ethyl linker coupled to Affi-Gel 10 was de-
signed and synthesized as described previously with modification
(Scheme 1).6–8 Then, in order to identify the proteins that bind to
the chemical affinity matrix, 2b was incubated with RAW264.7 cell
lysate and then the bound proteins were resolved by SDS–PAGE as
shown in Figure 2A. Arrow-indicated proteins (T1–T5) were iden-
tified by LCQ mass spectrometry peptide mass fingerprinting after
in-gel digestion as described previously (Table 1).9 Since among 5
candidate proteins binding to 2b, prohibitin had higher Mascot
score (446) than others, we further evaluated whether prohibitin
Figure 2. Identification of putative 2b-binding proteins in RAW264.7 cell lysate.
Putative 2b-binding proteins was identified in Commassie Blue-stained gel
compared to unbound (lane 1), subsequent washing fractions (lanes 2–4) and
boiled (or bound) fraction (lane 5). The left lane (M) was the standards of molecular
weight. (B) Prohibitin levels in unbound (Un) and bound (B) fractions of non-
conjugated Affi-Gel 10 beads and 2b were evaluated by western blot analysis. Same
amount of cell lysate were incubated with non-conjugated Affi-Gel 10 beads or 2b.
could interact with 2b by western blot analysis.10 As shown in
Figure 2B, prohibitin was strongly detected in the bound fraction
c
a, b
O
O
BocHN
H2N
OH
BocHN
OTs
O
2
3
4
5
BocHN
N
O
S
O
d
O
BocHN
2
+
+
O
S
N3
O
N
N
H
2
O
2a
5
6
7
Affi-Gel 10
f
agarose
R =
R
N
2
e
O
O
H
H
N
O
N
N
S
O
O
N
H
O
O
2b
Scheme 1. Synthesis of chemical affinity matrix 2b. The amino group of 2-(2-aminoethoxy)ethanol (3) was protected with Boc group, and then hydroxyl group was tosylated.
The resulting tosylated compound 4 was reacted with propargyl alcohol in the presence of NaH to give the linker part 5 containing terminal alkyne moiety. The terminal
alkyne compound 5, sulfonyl azide compound 6, and amine compound 7 gave sufonyl amidine compound 2a in the presence of Cu catalysis at room temperature in 57%
yield.6 After deprotecting of Boc group with trifluoroacetic acid, the resulting amino group was coupled with Affi-Gel in the presence of diisopropylethylamine to give the final
chemical affinity matrix 2b.7 Reagents and conditions: (a) (Boc)2O (1.0 equiv), Et3N (1.0 equiv), Dioxane, 0 °C to rt, 12 h, 99%; (b) TsCl (1.5 equiv), Et3N (3.0 equiv), CH2Cl2, 0 °C
to rt, 4 h, 85%; (c) 60% NaH (10 equiv), propargyl alcohol (10 equiv), THF, 0 °C to rt, 2 d, 82%; (d) CuI (0.2 equiv), THF, rt, 3 h, 57%. (e) TFA (15 equiv), CH2Cl2, rt 1 h, 99%; (f)
conjugation with Affi-Gel 10.7

S.-Y. Chang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 727–729
729
Table 1
Acknowledgment
Candidate proteins binding to 2b
No.
T1
Accession number
GI31543113
Protein name
MASCOT score
269
This work was supported by a grant from the Korea Healthcare
Technology R&D Project, Ministry of Health, Welfare & Family Af-
fairs, Republic of Korea (A084242).
Lymphocyte cytosolic
protein 1 (L-plastin)
Unnamed protein product
Gamma-actin
Glyceraldehyde 3-phosphate-
dehydrogenase
T2
T3
T4
GI55491
GI809561
GI56188
381
410
292
References and notes
1. Han, S.-Y.; Kim, S. H. Arch. Pharm. Chem. Life Sci. 2007, 340, 169.
2. Rix, U.; Superti-Furga, G. Nat. Chem. Biol. 2010, 5, 616.
T5
GI4505773
Prohibitin
446
3. Boyle, W. J.; Simonet, W. S.; Lacey, D. L. Nature 2003, 423, 337.
4. Harada, S.; Rodan, G. A. Nature 2003, 423, 349.
5. Lee, M. Y.; Kim, M. H.; Kim, J.; Kim, S. H.; Kim, B. T.; Jeong, I. H.; Chang, S.; Kim,
S. H.; Chang, S.-Y. Bioorg. Med. Chem. Lett. 2010, 20, 541.
6. (a) Bae, I.; Han, H.; Chang, S. J. Am. Chem. Soc. 2005, 127, 2038; (b) Cho, S. H.;
Yoo, E. J.; Bae, I.; Chang, S. J. Am. Chem. Soc. 2005, 127, 16046; (c) Yoo, E. J.; Bae,
I.; Cho, S. H.; Han, H.; Chang, S. Org. Lett. 2006, 8, 1347; (d) Chang, S.; Lee, M.;
Jung, D. Y.; Yoo, E. J.; Cho, S. H.; Han, S. K. J. Am. Chem. Soc. 2006, 128, 12366; (e)
Yoo, E. J.; Ahlquist, M.; Kim, S. H.; Bae, I.; Fokin, V. V.; Sharpless, K. B.; Chang, S.
Angew. Chem., Int. Ed. 2007, 46, 1730; (f) Cho, S. H.; Chang, S. Angew. Chem., Int.
Ed. 2007, 46, 1897; (g) Cho, S. H.; Chang, S. Angew. Chem., Int. Ed. 2008, 47, 2836;
(h) Yoo, E. J.; Chang, S. Org. Lett. 2008, 10, 1163; (i) Yoo, E. J.; Ahlquist, M.; Bae,
I.; Fokin, V. V.; Sharpless, K. B.; Chang, S. J. Org. Chem. 2008, 73, 5520.
7. Conjugation of 2a with Affi-Gel 10 was performed as the following; Affi-Gel 10
(0.5 ml; Bio-Rad) was transferred into a 3-ml cartridge with 20mPE FRIT
(Applied Separations). The supernatant solvent was drained and Affi-Gel 10
of 2b suggesting that prohibitin could be one of specific binding
proteins to 2b. Although L-plastin with low Mascot score was de-
tected in both the unbound and bound fraction of 2b, we could
not conclude that L-plastin is the unspecific binding protein to
2b because it still exhibited the binding potential to 2b. Binding
kinetics study based on surface plasmon resonance (SPR) would ac-
cess their binding potentials to 2b in a further study.
Prohibitin is ubiquitous, evolutionarily conserved protein that is
mainly localized and functions in mitochondria.11 The functional
role of prohibitin in development, senescence and tumor suppres-
sion has been also proposed, but there was no evidence to eluci-
date the functional involvement of prohibitin in the
osteoclastogenesis. However, its repressive role in the transcrip-
was washed with DMSO three times. After capping the cartridge with
a
stopper, a solution of 3.75 mol of 2a in 0.5 ml DMSO and 50 l of N,N-
l
l
diisopropylethylamine (DIEA) were added to the gel. After shaking for 3 h at
room temperature, the slurry was drained and the gel was washed with DMSO
three times. Then, 1 ml DMSO with 50 mM ethanolamine and 15 ll of DIEA
were added to the gel. After shaking for 3 h at room temperature, the slurry
was drained and the gel was washed with DMSO three times and saturated
with buffer A (25 mM Tris–HCl, pH 7.4, 0.25 M sucrose, 150 mM NaCl, 1 mM
CaCl2, 1 mM PMSF, 1% Nonidet P-40 and protease inhibitor cocktail).
8. Khersonsky, S. M.; Jung, D.-W.; Kang, T.-W.; Walsh, D. P.; Moon, H.-S.; Jo, H.;
Jacobson, E. M.; Shetty, V.; Neubert, T. A.; Chang, Y.-T. J. Am. Chem. Soc. 2003,
125, 11804.
9. Kim, M. H.; Choi, Y. L.; Heo, J.-N.; Min, Y. K.; Kim, S. H. Bull. Korean Chem. Soc.
2010, 31, 2047; Protein sample preparation and its incubation with 2b.
RAW264.7 cells were homogenized (1:5, w/v) in buffer A under sonication. The
homogenate was then centrifuged at 10,000g for 15 min and 0.5 ml of the
tional activity of estrogen receptor
a (ERa) made us suggest the
hypothesis that sulfonyl amidine derivatives with anti-resorptive
activities might lead to the activation of the estrogen signaling
by the inhibition of prohibitin to bind with estrogen receptor that
is consequently activated and suppress the osteoclastogenesis.12
Indeed, the osteoclastic ERa-mediated osteoprotective action of
estrogens has been reported in several studies. The functional
involvement of prohibitin in osteoclastogenesis and its relation-
ship with estrogen signaling as well as its binding kinetics with
2b would be accessed in a further study.
supernatant diluted with buffer
A (7 mg/ml) was stirred with buffer
A-saturated 2b at 4 °C for 15 h. After incubation, the resins were precipitated
by centrifugation at 10,000g for 1 min and washed three times with 1 ml of
The expression of L-plastin (also referred to L-fimbrin) that is
localized in membrane ruffles and cell adhesion sites has been
shown to be restricted to cells of the haematopoietic lineage
including macrophages, but the sequential expression and differ-
ential localization of fimbrin isoforms during epithelial cell differ-
entiation has been also reported.13 Interestingly, L-plastin, an
actin-bundling protein, has been shown to be a component of the
osteoclast adhesion complexes called podosomes that is rare in
the primarily mononucleated cells, but commonly found at the
periphery of multinucleated cells indicating that the number of
cells containing L-plastin-positive podosomes increased during
osteoclastogenesis.14 Therefore, the inhibition of L-plastin might
affect the formation and functional role of podosomes in the early
stage of osteoclastogenesis; in turn, it could block the formation of
multinucleated osteoclasts. After confirming the binding potential
of 2b to L-plastin, its functional involvement in the osteoclasto-
genesis might be evaluated.
In conclusion, we here designed and synthesized 2b as the
chemical affinity probe for the sulfonyl amidine derivatives with
anti-resorptive activity, and by using 2b-based chemical proteo-
mics, prohibitin was identified as one of binding proteins for 2b.
Further studies such as SPR-based binding kinetics, gene silencing
and X-ray co-crystallography could be required for validating the
interaction between bioactive small molecule and its binding pro-
teins, and elucidating the functional involvement of the binding
proteins in the biological event of interest.
buffer A. The washed beads were then resuspended with 20 ll of SDS sample
buffer (100 mM Tris–HCl, 2% sodium dodecyl sulfate, 1% 2-mercaptoethanol,
2% glycerol, 0.01% bromophenol blue, pH 7.6), incubated at 25 °C for 10 min,
and subjected to SDS–PAGE followed by the gel staining.
10. Western blot analysis. RAW264.7 cell lysate (40 lg) were incubated with non-
conjugated Affi-Gel 10 or 2b at 4 °C for 15 h and centrifuged at 10,000g for 1 min.
The supernatant (unbound fractions) was denatured with SDS sample buffer.
Then, the pellets were washed twice with buffer A, resuspended with 20 ll of
SDS sample buffer and incubated at 25 °C for 10 min for getting bound fractions.
Denaturation of unbound and bound fractions was done by boiling and then
samples were subjected to 10% polyacrylamide gels. Electrophoresis was
performed using the Mini Protean 3 Cell (Bio-Rad). The resolved proteins were
transferred to PVDF membrane (Millipore). The membranes were incubated in
blocking buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 3%
nonfat dry milk) and then incubated with diluted primary antibodies (1:1000)
for 2 h at room temperature. Antibodies used in this study were purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz). Following the primary antibody
reactions, the membranes were washed with blocking buffer three times
(15 min each) and then probed with diluted secondary antibodies (1:2000) for
1 h. The membranes were washed three times (15 min each) and developed with
SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology)
using the LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd).
11. (a) Artal-Sanz, M.; Tavernarakis, N. Trends Endocrinol. Metab. 2009, 20, 394; (b)
Osman, C.; Merkwirth, C.; Langer, T. J. Cell Sci. 2009, 122, 3823.
12. (a) García Palacios, V.; Robinson, L. J.; Borysenko, C. W.; Lehmann, T.; Kalla, S.
E.; Blair, H. C. J. Biol. Chem. 2005, 280, 13720; (b) Sørensen, M. G.; Henriksen, K.;
Dziegiel, M. H.; Tankó, L. B.; Karsdal, M. A. DNA Cell Biol. 2006, 25, 475; (c) Imai,
Y.; Kondoh, S.; Kouzmenko, A.; Kato, S. Mol. Endocrinol. 2010, 24, 877.
13. (a) Messier, J. M.; Shaw, L. M.; Chafel, M.; Matsudaira, P.; Mercurio, A. M. Cell
Motil. Cytoskeleton 1993, 25, 223; (b) Chafel, M. M.; Shen, W.; Matsudaira, P.
Dev. Dyn. 1995, 203, 141.
14. Babb, S. G.; Matsudaira, P.; Sato, M.; Correia, I.; Lim, S. S. Cell Motil. Cytoskeleton
1997, 37, 308.