Ridaifen B, a tamoxifen derivative, directly binds to Grb10 interacting GYF protein 2

Article abstract of DOI:10.1016/j.bmc.2012.10.037

Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.

Full text of DOI:10.1016/j.bmc.2012.10.037

Bioorganic & Medicinal Chemistry 21 (2013) 311–320
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ridaifen B, a tamoxifen derivative, directly binds to Grb10 interacting GYF
protein 2
Senko Tsukuda a, Tomoe Kusayanagi a, Eri Umeda b, Chihiro Watanabe b, Yu-ta Tosaki b, Shinji Kamisuki a,
Toshifumi Takeuchi a, Yoichi Takakusagi a, Isamu Shiina b, Fumio Sugawara a,
⇑
a Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was
reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer
cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for
some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage dis-
play screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as
a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was
found to have decreased in the presence of RID-B.
Received 31 August 2012
Revised 3 October 2012
Accepted 5 October 2012
Available online 29 October 2012
Keywords:
Ridaifen B
Tamoxifen
Phage display
GIGYF2
Ó 2012 Elsevier Ltd. All rights reserved.
Akt
1. Introduction
treatment including tamoxifen.9 These data indicated that there
may be an unknown ER-independent mode of action for RID-B.
Tamoxifen (Fig. 1) is an antagonist of the estrogen receptor (ER)
and is widely used in hormone therapy and to prevent the recur-
rence of cancer in patients with ER-positive breast cancers.1,2 In
breast cancer cells, tamoxifen inhibits cell proliferation by blocking
growth signals and inducing apoptosis.3 However, recent reports
have shown that tamoxifen induces apoptosis even in ER-negative
cells, which suggests that tamoxifen has other target proteins.4,5
Additionally, tamoxifen can induce some major side effects includ-
ing a higher incidence of endometrial cancer and drug resistance
following long-term therapy.6,7 In our previous studies we synthe-
sized novel tamoxifen derivatives in order to better understand the
mechanism of action of this drug and to generate new therapeutic
agents with reduced side effects.8,9
To identify RID-B-binding proteins, we used a T7 phage display
method. Phage display is a powerful technique for identifying pep-
tides or proteins that bind to proteins or small molecules of inter-
est. However, unlike protein samples, passive immobilization of
Ridaifen B (RID-B), one of these tamoxifen derivatives (Fig. 1),
showed a higher inhibitory activity on cellular proliferation than
that of tamoxifen. The global anti-tumor activity of RID-B against
a variety of human cancer cells showed that RID-B displayed no
difference in inhibitory activity between ER-positive and ER-nega-
tive cells.9,10 Furthermore, on an analysis using a COMPARE pro-
gram, it was suggested that the mode of action for RID-B was
different from that for more than 200 of existing drugs for cancer
⇑
Corresponding author. Tel.: +81 471 24 1501x3400; fax: +81 471 23 9767.
E-mail address: sugawara@rs.noda.tus.ac.jp (F. Sugawara).
Figure 1. Chemical structures of tamoxifen, RID-B and RID-D.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.037

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S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
small molecules onto a plate surface is generally unsuccessful.
Therefore, biotinylated small molecule derivatives and avidin-
coated microplates are universally used for affinity selection. A
T7 phage display method uses a library constructed from T7 phage
particles. Each phage particle expresses peptides of up to about
1200 amino acids in length on the capsid that is identical to pro-
teins or their fragments encoded in living cells or organs.11–13
Through several rounds of screening, phage particles displaying
amino acid sequences that recognize the small molecule of interest
are gradually enriched. The amino acid sequences expressed on the
capsid of the selected phage particles are then easily identified by
sequencing the phage DNA. A subsequent similarity search in the
genome database enables a prediction of the potential binding
partner(s) as well as the likely binding site. Moreover, unlike many
other methods, phage display does not rely on the target protein
being soluble and expressed at relatively high levels. Thus, phage
display facilitates the identification of less soluble proteins, such
as membrane proteins, as well as proteins with a low expression
level. In the past several years, we have determined a number of
binding partners of small molecules by using a T7 phage display
screening protocol.14–20
and GIGYF2. Furthermore, using a cell-based assay, the Akt phos-
phorylation level mediated by GIGYF2 was found to have de-
creased in the presence of RID-B. These results might explain a
possible ER-independent mode of action for RID-B.
2. Results and discussion
2.1. Synthesis of biotinylated RID-B and ridaifen D derivatives
Biotinylated RID-B (Bio-RID-B), 2-[3-(N-biotinyl-6-aminohexa-
noyl)phenyl]-1,1-bis{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-1-butene
(7), was synthesized by the Mukaiyama reductive coupling reac-
tion,23,24 and the Shiina esterification,25–27 as shown in Scheme 1.
First, 1-[3-(benzyloxy)phenyl]propanone (2)28 was treated with
an excess amount of 4,40-dihydroxybenzophenone (1) in the pres-
ence of the low-valent titanium species generated from tita-
nium(IV) chloride with zinc powder to afford the desired cross
coupling product 3 in 94% yield. The phenol moieties in 3 were
transformed into the corresponding aminoethyl ethers by alkyl-
ation in 88% yield, and the benzyl protective group in the tetra-
substituted olefin 4 was then cleaved under hydrogenation condi-
tions to provide the phenol derivative 5 in 92% yield. Finally, rapid
esterification of N-biotinyl-6-aminohexanoic acid (6) with the RID-
B derivative 5 was carried out in dimethylformamide (DMF) sol-
vent using 2-methyl-6-nitrobenzoic anhydride (MNBA) with 4-
(dimethylamino)pyridine (DMAP). The facile dehydration process
successfully furnished the probe molecule 7 in 70% yield. It is note-
worthy that this is the first application of the MNBA-mediated
Here we report the identification of Grb10 interacting GYF
protein 2 (GIGYF2) as a RID-B-binding protein using a T7 phage
display screen. It has been demonstrated that GIGYF2 regulates
phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway of
the receptor tyrosine kinase, which is involved in cell prolifera-
tion.21,21 A bead pull-down assay and surface plasmon resonance
(SPR) analyses confirmed the direct interaction between RID-B
Scheme 1. Synthesis of a biotinylated RID-B derivative (Bio-RID-B) (7).

S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
313
Scheme 2. Synthesis of a biotinylated RID-D derivative (Bio-RID-D) (10).
coupling reaction between carboxylic acids and alcohols to the
expeditious and effective synthesis of the biotin labeled small
molecules.29,30
Next, biotinylated RID-D (Bio-RID-D), 2-[3-(N-biotinyl-6-ami-
nohexanoyl)phenyl]-1,1-bis{4-[2-(morpholin-4-yl)eth-
oxy]phenyl}-1-butene (10), was also synthesized using a similar
procedure as described in Scheme 2. Aminoethyl groups were
introduced into the bisphenol 3 in 96% yield, and the phenol deriv-
ative 9 was successively prepared from the benzyl ether 8 by
hydrogenation in 80% yield. Facile esterification of 6 with the
RID-D derivative 9 also took place in the presence of MNBA and
DMAP to afford the probe molecule 10 in 72% yield.
2.2. Selection of RID-B-binding protein using a T7 phage display
screen
To explore RID-B-binding proteins, we performed a phage dis-
play screen. A T7 phage library was constructed from a cDNA li-
brary of Jurkat cells, an estrogen receptor-negative human
leukemia cell line. First, the T7 phage library was incubated with
Bio-RID-B immobilized on streptavidin-coated wells. After incuba-
tion, unbound and non-specifically bound phage particles were
removed by washing. Finally, the remaining phage particles on
the wells were eluted and amplified for the next round. The ratio
of recovered phage titer dramatically increased after four rounds
of selection, indicating the enrichment of T7 phage particles that
potentially bind to RID-B (Fig. 2A). Fifteen individual phage plaques
were randomly isolated after the fourth round of selection and the
amino acid sequence of the displayed protein was determined.
Three out of the 15 phage clones had the same DNA sequence
encoding a polypeptide of 286 a.a., whereas the remaining 12
phage clones encoded a polypeptide of 135 a.a. (Table 1). We also
Figure 2. Selection of RID-B-binding protein by a T7 phage display screening
procedure. (A) Relative enrichment of T7 phage particles binding to RID-B in each
round of selection. The phage titer in the eluate from the Bio-RID-B-immobilized
well was compared to the input library titer and shown as recovery ratio. (B)
Affinity check of the 286 a.a. phage clone for RID-B. The T7 phage that displays the
286 a.a. [peptide display (+)] or control phage clone [peptide display (ꢀ)] was
incubated in a well with [RID-B (+)] or without [RID-B (ꢀ)] of Bio-RID-B and shown
as recovery ratio (%). (C) Schematic representation of full-length GIGYF2. Underline
indicates the region that corresponds to the 286 a.a. sequence displayed on the RID-
B-binding T7 phage clone.
performed the screening of clones that bind to ridaifen D (RID-D) (
Fig. 1), whose cytotoxic activities are lower than that of RID-B, as
well as a screen for clones that bind nonspecifically to the control
well containing no immobilized small molecule. We obtained two

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S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
Table 1
Amino acid sequences and frequency of the phage clones selected from the Bio-RID-B immobilized well
Amino acid sequence
Number of amino acid
135
Frequency
12/15
DPSKGRWVEGITSEGYHYYYDLISGASQWEKPEGFQGDLKKTAVKTVWVEGLSEDGFTYYYNTETGE
SRWEKPDDFIPHTSDLPSSKVNENSLGTLDESKSSDSHSDSDGEQEAEEGGVSTETEKPKIKFKEKNK
EMRAKREEEERKRQEELRRQQEEILRRQQEEERKRREEEELARRKQEEALRRQREQEIALRRQREEEERQQQEEA
LRRLEERRREEEERRKQEELLRKQEEEAAKWAREEEEAQRRLEENRLRMEEEAARLRHEEEERKRKELEVQR
QKELMRQRQQQQEALRRLQQQQQQQQLAQMKLPSSSTWGQQSNTTAEMRAKREEEERKRQEELRRQQE
EILRRQQEEERKRREEEELARRKQEEALRRQREQEIALRRQREEEERQQQEEALRRLEERRREEEERRK
286
3/15
After the fourth-round of selection by T7 phage display, 15 single phage clones were randomly picked and their DNA sequences analyzed to determine the phage displayed
peptides.
clones from the Bio-RID-D-immobilized well and two clones from
the control well. The phage clone displaying a polypeptide of 135
a.a. was observed in the eluate from the control well in addition
to the RID-B and RID-D immobilized wells, suggesting this was a
nonspecific binding phage clone (Tables 1, S1 and S2).
RID-B in a dose-dependent manner and global fitting gave a KD va-
lue of 186 nM. These results indicate that RID-B strongly interacts
with the 745–1030 a.a. region in GIGYF2 (Fig. 3D).
We also compared the affinity of the ridaifen analogs for GI-
GYF2. Previously, we synthesized several ridaifen derivatives and
found that the cytotoxic activity of the ridaifen derivatives was
We decided to check the affinity of the T7 phage displaying the
286 a.a. polypeptide for RID-B. The corresponding T7 phage clone
and a control T7 phage clone displaying no peptide were amplified
and then separately added to the RID-B-immobilized well or a
blank control well. The bound T7 phage particles were then recov-
ered and the ratio of the two phage clones isolated from the RID-B-
immobilized well was compared to that from the non-immobilized
control well. The recovery ratio of the phage clone displaying the
286 a.a. polypeptide was four-fold higher from the RID-B immobi-
lized well than from the blank control well. Furthermore, the con-
trol phage clone did not bind to RID-B. These results indicated that
the phage clone displaying the 286 a.a. polypeptide selectively
interacts with RID-B (Fig. 2B).
A similarity search using FASTA identified that the 286 a.a.
sequence displayed on the RID-B-binding phage clone is identical
to the 745–1030 region of Grb10 interacting GYF protein 2 (GI-
GYF2) (Figs. 2C and S1). It has been reported that GIGYF2 is a
protein that interacts with growth factor receptor-bound protein
10 (Grb10) and has a critical role in the PI3K/Akt signaling path-
way. However, its molecular function is not fully understood.
Despite the high molecular weight of GIGYF2 (1299 a.a.), use
of T7 phage display and subsequent similarity search enabled
the identification of this protein as a potential RID-B-binding
partner along with a candidate binding site in 745–1030. This
286 a.a. sequence did not show any similarity with GIGYF1, an
another subtype of GIGYF, indicating that binding of RID-B to GI-
GYF is specific for subtype2.
dependent on its side chain.8,9 Among them, RID-D (LogGI50
=
ꢀ4.84 M) containing morpholine side chains showed low cytotox-
icity with non-apoptotic activity in contrast to RID-B (LogGI50
=
ꢀ5.93 M). In addition, no phage clone displaying GIGYF2 was ac-
quired from a T7 phage display screen using Bio-RID-D (Table
S1). Therefore, we speculated that the binding affinity between GI-
GYF2 and RID-D was less than that for RID-B. As shown in Figure
3B, we attempted the pull-down assay using Bio-RID-D and found
that Bio-RID-B-immobilized resin bound to Flag-tagged GIGYF2
more strongly than Bio-RID-D-immobilized resin. Furthermore,
SPR analysis showed that RID-B binds to GIGYF2(745–1030) at
the range of concentrations tested (Fig. 3D and E). By contrast, little
response was observed for RID-D, which is consistent with the re-
sults from the pull-down assay (Fig. 3F). Thus, the affinity of each
ridaifen derivative for GIGYF2 correlated with the cytotoxicity
data.
2.4. Effect of RID-B on the PI3K/Akt signaling pathway
Previous reports indicated that GIGYF2 was a candidate gene for
PARK11-linked Parkinson’s disease. Furthermore, recent studies
have reported the involvement of GIGYF2 in the PI3K/Akt signaling
pathway,21,22,31 and GIGYF2 was shown to be up-regulated in
breast cancer cells in which high phosphorylation levels of Akt
are observed. Thus we reasoned that the binding of RID-B to GI-
GYF2 might be biologically relevant. Initially, we examined
whether RID-B affects the phosphorylation level of Akt. HEK293T
cells transiently-expressing GIGYF2 were cultured in serum-free
medium following pretreatment with RID-B for 4 h, and then stim-
ulated with IGF-1 at different times. The phosphorylation of Akt at
Ser473, which is known to be a representative marker for activa-
tion of Akt,32–34 was detected by Western blot analysis using
anti-phospho-Akt (Ser473) antibodies. As shown in Figure 4, the
phosphorylation level of Akt at Ser473 15 min after IGF-1 stimula-
tion was significantly reduced by RID-B in a dose-dependent man-
ner. By contrast, there was no reduction in the phosphorylation
level of Akt when the experiment was conducted using cells trans-
fected with mock empty vector.
Taken together, our results demonstrate that RID-B directly binds
to GIGYF2 and reduces the phosphorylation level of Akt at Ser473.
This is the first report of a small molecule that binds to GIGYF2 to in-
hibit its function. Indeed, the knockdown of GIGYF2 reduced cell
proliferation in breast cancer cell lines,31 the RID-B-GIGYF2 interac-
tion and subsequent inhibitory effect of the Akt phosphorylation
may be relevant to the anti-proliferative activity of RID-B as well.
Further experiments will clarify the detailed mode of actions.
2.3. Interaction analysis between RID-B and GIGYF2
To confirm the interaction between GIGYF2 and RID-B, we per-
formed a pull-down assay. Flag-tagged GIGYF2 was transiently ex-
pressed in HEK-293T cells (Fig. 3A), and the cellular lysates were
incubated with Bio-RID-B- or biotin-immobilized resin. The bound
proteins were then analyzed by Western blotting using an
antibody against GIGYF2. Flag-tagged GIGYF2 was found to bind
to Bio-RID-B-immobilized resin, but not to the control resin
(Fig. 3B). We further elucidated the interaction and estimated the
dissociation constant (KD) between GIGYF2 and RID-B using sur-
face plasmon resonance (SPR) analysis. We engineered a His-
tagged truncated form of GIGYF2 corresponding to the 745–1030
region of GIGYF2 (GIGYF2(745–1030)) that is, the region displayed
on the RID-B-binding phage (Fig. 3A and C). The recombinant pro-
tein was then purified to homogeneity. Various concentrations of
GIGYF2(745–1030) were injected over the surface of the SA sensor
chip on which Bio-RID-B had been immobilized through the inter-
action between biotin and avidin. GIGYF2(745–1030) bound to

S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
315
Figure 3. Direct interaction of RID-B with GIGYF2. (A) Schematic representation of the engineered full length or truncated GIGYF2 protein. (B) Interaction of RID-B or
RID-D with Flag-tagged GIGYF2. Bio-RID-B, Bio-RID-D or biotin was fixed onto avidin beads and then incubated with cell extracts containing GIGYF2. After extensive
washing, the bound GIGYF2 was detected by Western blot analysis. (C) Purified recombinant GIGYF2(745–1030). Recombinant GIGYF2(745–1030) from a bacterial
expression system was purified to apparent homogeneity. The purity of the GIGYF2(745–1030) preparation was confirmed by SDS–PAGE analysis followed by staining
with CBB. (D–F) A sensorgram obtained from SPR analysis between biotinylated ridaifen derivative and GIGYF2(745–1030). Bio-RID-B (D, E) or Bio-RID-D (F) was
immobilized on a SA sensor chip. Solutions of purified GIGYF2(745–1030) at various concentrations (from top to bottom: 5.0, 4.0, 2.0, 1.0, 0.5 or 0.25 lM) was injected
over the surface of the sensor chip. The binding responses (in RU) were recorded as a function of time (in sec). The estimated KD value of interaction between RID-B and
GIGYF2(745–1030) was 186 nM.
4. Materials and methods
4.1. Chemistry
All reagents were purchased from Tokyo Kasei Kogyo Co., Ltd
(TCI, Tokyo, Japan), Kanto Chemical Co., Inc. (Tokyo, Japan) or Sig-
ma–Aldrich (St. Louis, MO), and used without further purification.
1-[3-(Benzyloxy)phenyl]propanone (2) was prepared from 3-ben-
zyloxybenzaldehyde (Sigma–Aldrich) according to the literature
Figure 4. Effect of RID-B on the Akt phosphorylation in GIGYF2-overexpressing
cells. HEK293T cells were transiently transfected with mock empty vector or Flag-
GIGYF2 expression vector, and were serum-depleted for 20 h. Cells were then
method.35–37 2-Methyl-6-nitrobenzoic anhydride (MNBA) was
purchased from Tokyo Kasei Kogyo Co., Ltd (M1439).
All melting points are uncorrected. 1H and 13C NMR spectra
treated with 0, 0.3 or 3
lM of RID-B for 4 h followed by stimulation with IGF-1 for 0
or 15 min. Each protein was detected by Western blot analysis using a specific
were recorded with chloroform (in chloroform-d) or methanol (in
methanol-d4) as an internal standard. Column chromatography
was performed on silica gel 60 (Merck, Darmstadt, Germany). Thin
layer chromatography was performed on Wakogel B5F. Unless
otherwise stated, all reactions were carried out under an atmo-
sphere of argon using dried glassware. Ammoniacal chloroform
was prepared from 28% aqueous ammonia by ammonia extraction
with chloroform.
antibody. The data is
experiments.
a representative example from three independent
3. Conclusion
Our investigations have identified a small molecule that tar-
gets GIGYF2, which is involved in the PI3K/Akt signaling path-
way and is up-regulated in breast cancer cells where Akt is
highly phosphorylated. We employed a T7 phage display screen
to identify RID-B-binding protein, a derivative of tamoxifen. Pull-
down assay and SPR analysis confirmed the selective interaction
between RID-B and GIGYF2. We also demonstrated that RID-B
reduces the phosphorylation level of Akt in cells overexpressing
GIGYF2. Taken together, our results show that RID-B directly
binds to GIGYF2 and reduces the phosphorylation level of Akt
at Ser473. Thus, binding of RID-B to GIGYF2 and subsequent
reduction of Akt phosphorylation might explain the ER-indepen-
dent anti-breast cancer activity of RID-B. Furthermore, RID-B and
its analogs, as well as being valuable tools for investigating the
molecular function of GIGYF2, could be important in the devel-
opment of novel anti-breast cancer chemotherapy drugs.
4.2. Compounds
RID-B was synthesized chemically as described in previous
reports.8,9
4.3. Antibodies
The following primary antibodies were used: rabbit polyclonal
anti-GIGYF2 (H-95, Santa Cruz Biotechnology, Santa Cruz, CA),
mouse monoclonal anti-GAPDH (6C5, Santa Cruz Biotechnology),
rabbit polyclonal anti-Akt (Cell Signaling Technology, Tokyo, Ja-
pan), rabbit polyclonal anti-phospho-Akt (Ser473; Cell Signaling
Technology).

316
S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
4.4. Synthesis of Bio-RID-B and Bio-RID-D
chloroform/methanol = 9:1) to afford 4 (267 mg, 88%) as a pale
yellow oil: IR (neat): 2962, 1604, 1512, 1242, 1041 cmꢀ1 1H
;
NMR (CDCl3): d 7.40–7.28 (5H, m, Ar), 7.15–7.04 (3H, m, Ar),
6.91–6.70 (7H, m, Ar), 6.59–6.55 (2H, m, Ar), 4.88 (2H, s, Bn),
4.12 (2H, t, J = 6.0 Hz, OCH2), 3.97 (2H, t, J = 6.0 Hz, OCH2), 2.91
(2H, t, J = 6.0 Hz, NCH2), 2.81 (2H, t, J = 6.0 Hz, NCH2), 2.66–2.54
(8H, m, pyrrolidinyl 2-H), 2.46 (2H, q, J = 7.2 Hz, 3-H), 1.84–
1.75 (8H, m, pyrrolidinyl 3-H), 0.93 (3H, t, J = 7.2 Hz, 4-H); 13C
NMR (CDCl3): d 158.3, 157.5, 156.7, 144.0, 140.7, 137.9, 137.1
(Ar), 136.2 (1), 135.8 (2), 131.7, 130.5, 129.6, 128.7, 128.4,
127.7, 127.4, 122.5, 116.3, 114.4, 114.0, 113.3, 112.7 (Ar), 69.8
(Bn), 66.9, 66.7 (OCH2), 55.05, 55.00 (pyrrolidinyl 2-C), 54.64,
54.59 (NCH2), 28.8 (3), 23.42, 23.38 (pyrrolidinyl 3-C), 13.6 (4);
HR MS: calcd for C41H48N2O3Na (M+Na+) m/z 639.3557, found
639.3585.
OH
OBn
HO
3
4.4.1. 2-[3-(Benzyloxy)phenyl]-1,1-bis(4-hydroxyphenyl)-1-
butene (3)
To a suspension of zinc powder (15.8 g, 242 mmol) in THF
(30 mL) at ꢀ10 °C was added titanium(IV) chloride (12.0 mL,
109 mmol). The reaction mixture was diluted with THF (20 mL)
and refluxed at 90 °C (bath temperature) for 2 h and then a mix-
ture of 4,40-dihydroxybenzophenone (1) (12.3 g, 57.3 mmol) and
1-[3-(benzyloxy)phenyl]propanone (2) (4.30 g, 17.9 mmol) in
THF (60 mL) was added to the mixture at 0 °C. After the reaction
mixture had been refluxed at 90 °C (bath temperature) for 2 h in
the dark, 10% aqueous potassium carbonate was added to the
mixture at 0 °C in the light. The mixture was filtered through
a short pad of Celite with ethyl acetate, and the filtrate was ex-
tracted with ethyl acetate. The organic layer was washed with
brine, and dried over sodium sulfate. After filtration of the mix-
ture and evaporation of the solvent, the crude product was puri-
fied by column chromatography on silica gel (eluant; hexane/
ethyl acetate = 2:1) to afford 3 (7.09 g, 94%) as a colorless solid:
mp: 153–156 °C; IR (KBr): 3402, 3039, 2962, 1589, 1504,
N
O
OH
N
O
5
4.4.3. 2-(3-Hydroxyphenyl)-1,1-bis{4-[2-(pyrrolidin-1-yl)ethoxy]
phenyl}-1-butene (5)
To a solution of 4 (109 mg, 0.177 mmol) in ethyl acetate
(5.89 mL) at room temperature under an atmosphere of argon
was added palladium on carbon (10% loading, 75.2 mg,
1234 cmꢀ1 1H NMR (CD3OD): d 7.36–7.26 (5H, m, Ar), 7.08–
;
6.95 (1H, m, Ar), 7.02 (2H, d, J = 8.5 Hz, Ar), 6.76 (2H, d,
J = 8.5 Hz, Ar), 6.73–6.67 (3H, m, Ar), 6.67 (2H, d, J = 9.0 Hz, Ar),
6.45 (2H, d, J = 9.0 Hz, Ar), 4.86 (2H, s, Bn), 2.46 (2H, q,
J = 7.5 Hz, 3-H), 0.90 (3H, t, J = 7.5 Hz, 4-H); 13C NMR (CD3OD):
d 159.8, 157.2, 156.4, 145.6, 141.3, 140.1, 138.8 (Ar), 136.4 (1),
136.3 (2), 132.9, 131.6, 130.7, 129.8, 129.4, 128.7, 128.6, 123.5,
117.9, 116.1, 115.8, 115.2, 114.1 (Ar), 71.0 (Bn), 29.7 (3), 14.0
(4); HR MS: calcd for C29H26O3Na (M+Na+) m/z 445.1774, found
445.1780.
70.7 lmol). The reaction mixture was stirred for 4.5 h at room
temperature under an atmosphere of hydrogen (1.0 atm) in the
dark, and then transferred to an atmosphere of argon in the
light. After filtration of the mixture through a short pad of
Celite with ethyl acetate and evaporation of the solvent, the
crude product was purified by thin layer chromatography on sil-
ica (eluant; ammoniacal chloroform/methanol = 9:1) to afford 5
(93.1 mg, 92%) as a pale yellow oil: IR (neat): 2970, 1604,
1512, 1242, 1049 cmꢀ1
; d 7.08 (2H, d,
1H NMR (CDCl3):
J = 8.5 Hz, Ar), 6.96 (1H, dd, J = 8.0, 8.5 Hz, Ar), 6.84 (2H, d,
J = 8.5 Hz, Ar), 6.76 (2H, d, J = 8.5 Hz, Ar), 6.60 (1H, d, J = 7.5 Hz,
Ar), 6.53 (2H, dd, J = 2.0, 4.0 Hz, Ar), 6.47 (2H, d, J = 8.5 Hz, Ar),
4.12 (2H, t, J = 6.0 Hz, OCH2), 3.91 (2H, t, J = 6.5 Hz, OCH2), 2.93
(2H, t, J = 6.0 Hz, NCH2), 2.80 (2H, t, J = 6.5 Hz, NCH2), 2.69–2.62
(4H, m, pyrrolidinyl 2-H), 2.62–2.55 (4H, m, pyrrolidinyl 2-H),
2.39 (2H, q, J = 7.5 Hz, 3-H), 1.85–1.72 (8H, m, pyrrolidinyl 3-
H), 0.87 (3H, t, J = 7.5 Hz, 4-H); 13C NMR (CDCl3): d 157.3,
156.7, 156.5, 144.1, 141.0, 137.3 (Ar), 136.4 (1), 135.9 (2),
131.8, 130.6, 128.9, 121.0, 116.9, 113.9, 113.6, 113.2 (Ar), 66.5,
65.9 (OCH2), 55.0, 54.9 (pyrrolidinyl 2-C), 54.6, 54.4 (NCH2),
29.2 (3), 23.3, 23.2 (pyrrolidinyl 3-C), 13.6 (4); HR MS: calcd
for C34H43N2O3 (M+H+) m/z 527.3268, found 527.3247.
N
O
OBn
N
O
4
4.4.2. 2-[3-(Benzyloxy)phenyl]-1,1-bis{4-[2-(pyrrolidin-1-yl)
ethoxy]phenyl}-1-butene (4)
To a solution of 3 (208 mg, 0.493 mmol) in DMF (4.93 mL) at
0 °C was added 60% sodium hydride (dispersion in paraffin
liquid, 118 mg, 2.96 mmol). The reaction mixture was stirred
for 15 min at 50 °C and then 1-(2-chloroethyl)pyrrolidine hydro-
chloride (278 mg, 1.63 mmol) was added in portions at room
temperature. After the reaction mixture had been stirred for
3 h at 50 °C, saturated aqueous ammonium chloride was added
at 0 °C. The mixture was extracted with dichloromethane, and
the organic layer was washed with water and brine, and then
dried over sodium sulfate. After filtration of the mixture and
evaporation of the solvent, the crude product was purified by
column chromatography on silica (eluant; ammoniacal
N
O
O
HN
H
NH
H
O
H
N
O
S
N
O
O
N-Biotinyl-6-aminohexanoic Acid Ridaifen B Ester (Bio-RID-B) (7)

S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
317
nyl OCH2), 3.69 (4H, t, J = 4.5 Hz, morpholinyl OCH2), 2.82 (2H, t,
J = 5.5 Hz, NCH2), 2.72 (2H, t, J = 5.5 Hz, NCH2), 2.59 (4H, t,
J = 4.5 Hz, morpholinyl NCH2), 2.52 (4H, t, J = 4.5 Hz, morpholinyl
4.4.4. 2-[3-(N-Biotinyl-6-aminohexanoyl)phenyl]-1,1-bis{4-[2-
(pyrrolidin-1-yl)ethoxy]phenyl}-1-butene (Bio-RID-B) (7)
To a solution of N-biotinyl-6-aminohexanoic acid (6) (20.0 mg,
NCH2), 2.45 (2H, q, J = 7.5 Hz, 3-H), 0.93 (3H, t, J = 7.5 Hz, 4-H); 13
C
56.0
67.1
11.2
l
l
l
mol), 2-methyl-6-nitrobenzoic anhydride (MNBA) (23.1 mg,
mol), 4-(dimethylamino)pyridine (DMAP) (1.37 mg,
mol) and triethylamine (47.0 L, 0.337 mmol) in dimethyl-
NMR (CDCl3): d 158.4, 157.4, 156.7, 144.0, 140.9, 137.9, 137.1 (Ar),
136.4 (1), 136.0 (2), 131.8, 130.6, 128.8, 128.5, 127.8, 127.5, 122.6,
116.4, 114.1, 113.4, 112.8 (Ar), 70.0 (Bn), 66.94, 66.89 (OCH2),
65.7, 65.5 (morpholinyl 2-C), 57.72, 57.67 (morpholinyl 3-C),
54.10, 54.07 (NCH2), 28.9 (3), 13.6 (4); HR MS: calcd for C41H48N2O5-
Na (M+Na+) m/z 671.3455, found 671.3489.
l
formamide (DMF) (1.41 mL) at 0 °C was added a solution of 5
(35.4 mg, 67.2 mol) in DMF (0.48 mL). After the reaction mixture
l
had been stirred for 30 min at room temperature, it was concen-
trated by evaporation of the solvent and then the residue was puri-
fied by thin layer chromatography on silica (eluant; ammoniacal
chloroform/methanol = 9:1) to afford 7 (33.9 mg, 70%) as a pale
yellow oil: IR (neat): 3302, 2931, 1759, 1705, 1643, 1512, 1242,
O
N
O
1049 cmꢀ1 1H NMR (CDCl3): d 7.14–7.10 (3H, m, Ar), 6.92–6.86
;
(4H, m, Ar), 6.82 (1H, dd, J = 2.0, 8.0 Hz, Ar), 6.76 (2H, d,
J = 9.0 Hz, Ar), 6.57 (2H, d, J = 9.0 Hz, Ar), 5.95–5.88 (1H, br m, 700-
NH), 5.83–5.72 (1H, br m, 800-NH), 5.07–5.00 (1H, br m, 60-NH),
4.48 (1H, dd, J = 5.5, 8.0 Hz, 800-H), 4.30 (1H, dd, J = 4.5, 8.0 Hz, 700-
H), 4.12 (2H, t, J = 6.0 Hz, OCH2), 3.98 (2H, t, J = 6.0 Hz, OCH2),
3.28–3.25 (2H, m, 60-H), 3.16–3.12 (1H, m, 600-H), 2.91 (2H, t,
J = 6.0 Hz, NCH2), 2.89 (1H, dd, J = 5.5, 13.0 Hz, 900-H), 2.82 (2H, t,
J = 6.0 Hz, NCH2), 2.71 (1H, d, J = 13.0 Hz, 900-H), 2.67–2.61 (4H, m,
pyrrolidinyl 2-H), 2.61–2.54 (4H, m, pyrrolidinyl 2-H), 2.51 (2H,
t, J = 7.0 Hz, 20-H), 2.46 (2H, q, J = 7.5 Hz, 3-H), 2.24–2.15 (2H, m,
200-H), 1.85–1.62 (14H, m, pyrrolidinyl 3-H, 30-H, 300-H, 500-H),
1.58–1.52 (2H, m, 50-H), 1.47–1.38 (4H, m, 40-H, 400-H), 0.93 (3H,
O
OH
N
O
9
4.4.6. 2-(3-Hydroxyphenyl)-1,1-bis{4-[2-(morpholin-4-yl)
ethoxy]phenyl}-1-butene (9)
To a solution of 8 (734 mg, 1.13 mmol) in ethyl acetate (32.0 mL)
at room temperature under an atmosphere of argon was added
palladium on carbon (10% loading, 532 mg, 0.500 mmol). The reac-
tion mixture was stirred for 48 h at room temperature under an
atmosphere of hydrogen (1.0 atm) in the dark, and then transferred
to an atmosphere of argon in the light. After filtration of the mixture
through a short pad of Celite with ethyl acetate and evaporation of
the solvent, the residue was diluted with ethyl acetate (32.0 mL) at
room temperature under an atmosphere of argon. Palladium on car-
bon (10% loading, 269 mg, 0.253 mmol) was added to the reaction
mixture, which was then stirred for 48 h at room temperature under
an atmosphere of hydrogen (1.0 atm) in the dark. The reaction mix-
ture was subsequently transferred to an atmosphere of argon in the
light. After filtration of the mixture through a short pad of Celite
with ethyl acetate and evaporation of the solvent, the crude product
was purified by column chromatography on silica (eluant; chloro-
form/methanol = 18:1) to afford 9 (507 mg, 80%) as a colorless solid:
13
t, J = 7.5 Hz, 4-H); C NMR (CDCl3): d 173.1 (100), 172.0 (10), 163.9
(urea), 157.6, 156.9, 150.4, 144.3, 139.7, 138.6 (Ar), 136.0 (1),
135.4 (2), 131.8, 130.4, 128.6, 127.5, 122.3, 119.0, 114.0, 113.5
(Ar), 67.0, 66.8 (OCH2), 61.7 (700), 60.1 (800), 55.6 (600), 55.10, 55.07
(pyrrolidinyl 2-C), 54.7, 54.6 (NCH2), 40.5 (900), 39.2 (60), 36.0 (200),
34.1 (20), 29.2 (50), 28.9 (3), 28.2 (400), 28.1 (500), 26.3 (40), 25.7
(300), 24.4 (30), 23.45, 23.43 (pyrrolidinyl 3-C), 13.6 (4); HR MS:
calcd for C50H68N5O6S (M+H+) m/z 866.4885, found 866.4842.
O
N
O
mp: 51–52 °C; IR (neat): 2959, 1605, 1509, 1243, 1035 cmꢀ1 1H
;
O
OBn
NMR (CDCl3): d 7.11 (2H, d, J = 8.5 Hz, Ar), 7.03 (1H, t, J = 8.0 Hz,
Ar), 6.87 (2H, d, J = 10.0 Hz, Ar), 6.77 (2H, d, J = 8.5 Hz, Ar), 6.67
(1H, d, J = 7.5 Hz, Ar), 6.58–6.53 (4H, m, Ar), 4.13 (2H, t, J = 5.5 Hz,
OCH2), 3.97 (2H, t, J = 5.5 Hz, OCH2), 3.74 (4H, t, J = 4.5 Hz, morpholi-
nyl OCH2), 3.70 (4H, t, J = 4.5 Hz, morpholinyl OCH2), 2.82 (2H, t,
J = 5.5 Hz, NCH2), 2.71 (2H, t, J = 5.5 Hz, NCH2), 2.60 (4H, t,
J = 4.5 Hz, morpholinyl NCH2), 2.53 (4H, t, J = 4.5 Hz, morpholinyl
N
O
4.4.5. 2-[3-(Benzyloxy)phenyl]-81,1-bis{4-[2-(morpholin-4-yl)
ethoxy]phenyl}-1-butene (8)
A dispersion of sodium hydride in paraffin liquid (60%, 287 mg,
7.17 mmol) was washed with petroleum ether under an atmo-
sphere of argon. A solution of 3 (501 mg, 1.18 mmol) in DMF
(12.0 mL) was then added to the resulting sodium hydride at 0 °C.
The reaction mixture was stirred for 15 min at room temperature
and then 4-(2-chloroethyl)morpholin hydrochloride (724 mg,
3.89 mmol) was added to the suspension in portions at room
temperature. After the reaction mixture had been stirred for 6 h at
50 °C, saturated aqueous ammonium chloride was added at 0 °C.
The mixture was extracted with dichloromethane, and the organic
layer was washed with water and brine, and dried over sodium sul-
fate. After filtration of the mixture and evaporation of the solvent,
the crude product was purified by column chromatography on silica
(eluant; chloroform/methanol = 20:1) to afford 8 (734 mg, 96%) as a
NCH2), 2.43 (2H, q, J = 7.5 Hz, 3-H), 0.92 (3H, t, J = 7.5 Hz, 4-H); 13
C
NMR (CDCl3): d 157.4, 156.7, 155.3, 144.4, 140.7, 137.8 (Ar), 136.4
(1), 135.9 (2), 131.8, 130.6, 129.0, 122.2, 116.7, 114.1, 113.4, 113.1
(Ar), 66.9, 66.8 (OCH2), 65.7, 65.3 (morpholinyl 2-C), 57.73, 57.65
(morpholinyl 3-C), 54.1, 54.0 (NCH2), 29.1 (3), 13.6 (4); HR MS: calcd
for C34H43N2O5 (M+H+) m/z 559.3166, found 559.3148: calcd for
C
34H42N2O5Na (M+Na+) m/z 581.2986, found 581.2978.
O
N
O
O
S
pale yellow oil: IR (neat): 2958, 1603, 1507, 1243, 1033 cmꢀ1 1H
;
HN
H
NH
H
O
H
N
NMR (CDCl3): d 7.37 (4H, d, J = 3.5 Hz, Ar), 7.33–7.30 (1H, m, Ar),
7.13 (2H, d, J = 8.5 Hz, Ar), 7.07 (1H, t, J = 8.0 Hz, Ar), 6.88 (2H, d,
J = 8.5 Hz, Ar), 6.77 (2H, d, J = 9.0 Hz, Ar), 6.75–6.71 (3H, m, Ar),
6.56 (2H, d, J = 8.5 Hz, Ar), 4.89 (2H, s, Bn), 4.13 (2H, t, J = 5.5 Hz,
OCH2), 3.98 (2H, t, J = 5.5 Hz, OCH2), 3.75 (4H, t, J = 4.5 Hz, morpholi-
O
O
N
O
O
N-Biotinyl-6-aminohexanoic Acid Ridaifen D Ester (Bio-RID-D) (10)

318
S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
4.6. Similarity search
4.4.7. 2-[3-(N-Biotinyl-6-aminohexanoyl)phenyl]-1,1-bis{4-[2-
(morpholin-4-yl)ethoxy]phenyl}-1-butene (Bio-RID-D) (10)
A similarity scores were obtained by FASTA (http://www.ebi.a-
c.uk/Tools/sss/fasta/).
To
(16.0 mg,
(MNBA) (18.5 mg, 53.7
(1.09 mg, 8.92 mol) and triethylamine (37.4
dimethylformamide (DMF) (1.34 mL) at 0 °C was added a solu-
tion of 9 (30.0 mg, 53.7 mol) in DMF (0.45 mL). After the reac-
a
solution of N-biotinyl-6-aminohexanoic acid (6)
44.8 mol), 2-methyl-6-nitrobenzoic anhydride
mol), 4-(dimethylamino)pyridine (DMAP)
L, 0.268 mmol) in
l
l
4.7. Construction of expression vectors
l
l
The phage clone displaying 745–1030 a.a. of GIFYF2 was used as
a template for the PCR. DNA encoding GIGYF2(745–1030) was
amplified using the following primer set: forward, 50-CATATGG
AAATGAGGGCAAAA-30, and reverse, 50-CTCGAGTCAACGAGCTCTG
TTTGGTTGCT-30 (underlined bases highlight the restriction endo-
nuclease recognition sites). The PCR product was inserted into
pET-28a (+) (Merck) expression vector in-frame with a His-tag at
the N-terminus. A full-length GIGYF2 cDNA clone was purchased
from Kazusa DNA Research Institute (Gene No. KIAA0642, Chiba,
Japan) and the plasmid DNA was used as template for the PCR.
The full-length GIGYF2 cDNA was constructed using the following
primer set: forward, 50-GCGGCCGCGGCAGCGGAAACGCAGACACT-30,
and reverse, 50-GGATCCTCAGTAGTCATCCAACGTCTCGATTTC-30. The
PCR product was inserted into p3xFLAG-CMV-10 (Sigma–Aldrich)
expression vector in-frame with 3ꢁ FLAG-tag at the N-terminus.
l
tion mixture had been stirred for 1.5 h at room temperature, it
was concentrated by evaporation of the solvent and then the
residue was purified by thin layer chromatography on silica (elu-
ant; ammoniacal chloroform/methanol = 9:1) to afford 10
(28.4 mg, 72%) as a pale yellow oil: IR (neat): 3303, 2927,
1765, 1708, 1643, 1507, 1243, 1039 cmꢀ1 1H NMR (CDCl3): d
;
7.13–7.10 (3H, m, Ar), 6.90–6.75 (7H, m, Ar), 6.55 (2H, d,
J = 9.0 Hz, Ar), 6.45–6.27 (1H, br m, 700-NH), 6.18–6.06 (1H, br
m, 800-NH), 5.53–5.36 (1H, br m, 60-NH), 4.47 (1H, dd, J = 4.5,
7.5 Hz, 800-H), 4.29 (1H, dd, J = 5.0, 7.0 Hz, 700-H), 4.12 (2H, t,
J = 5.5 Hz, OCH2), 3.97 (2H, t, J = 5.5 Hz, OCH2), 3.73 (4H, t,
J = 5.0 Hz, morpholinyl OCH2), 3.69 (4H, t, J = 5.0 Hz, morpholinyl
OCH2), 3.24 (2H, dd, J = 6.0, 7.5 Hz, 60-H), 3.13 (1H, dd, J = 4.5,
7.5 Hz, 600-H), 2.88 (1H, dd, J = 4.5, 13.5 Hz, 900-H), 2.81 (2H, t,
J = 5.5 Hz, NCH2), 2.72–2.69 (3H, m, NCH2, 900-H), 2.58 (4H, t,
J = 5.0 Hz, morpholinyl NCH2), 2.53–2.50 (6H, m, morpholinyl
NCH2, 20-H), 2.45 (2H, q, J = 8.0 Hz, 3-H), 2.19 (2H, t, J = 7.0 Hz,
200-H), 1.76–1.63 (6H, m, 30-H, 300-H, 500-H), 1.57–1.52 (2H, m,
50-H), 1.47–1.38 (4H, m, 40-H, 400-H), 0.93 (3H, t, J = 8.0 Hz, 4-
H); 13C NMR (CDCl3): d 173.1 (100), 172.0 (10), 163.8 (urea),
157.5, 156.8, 150.5, 144.2, 139.9, 138.5 (Ar), 136.1 (1), 135.5
(2), 131.8, 130.5, 128.7, 127.5, 122.3, 119.0, 114.1, 113.5 (Ar),
66.92, 66.87 (OCH2), 65.7, 65.6 (morpholinyl 2-C), 61.7 (700),
60.1 (800), 57.7, 57.6 (morpholinyl 3-C), 55.6 (600), 54.1, 54.0
(NCH2), 40.5 (900), 39.2 (60), 36.0 (200), 34.1 (20), 29.3 (50), 28.9
(3), 28.2 (400), 28.1 (500), 26.3 (40), 25.7 (300), 24.4 (30), 13.6 (4);
HR MS: calcd for C50H68N5O8S (M+H+) m/z 898.4783, found
898.4740: calcd for C50H67N5O8SNa (M+Na+) m/z 920.4603, found
920.4615.
4.8. Expression and purification of recombinant GIGYF2(745–
1030)
The engineered GIGYF2(745–1030) expression vector was used
to transform E. coli Rosetta 2 (DE3)plysS (Merck). These bacteria
were grown in LB medium containing 30
and 100 g/mL of chloramphenicol at 37 °C until the OD600 reached
0.5 before addition of 1 mM isopropyl thio-b- -galactoside (IPTG).
lg/mL of kanamycin
l
D
The cells were then incubated for a further 4 h at 37 °C. After incu-
bation, the cells were harvested and suspended in elution buffer I
(50 mM Na2HPO4, 5% glycerol, 0.05% Triton X-100, pH 7.2) contain-
ing protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The
cells were disrupted by sonication and the resulting extract was
clarified by centrifugation (17,500g) at 4 °C for 10 min. Solid
ammonium sulfate was then gradually added to the clarified cell-
free extract to a final concentration of 40% mass/volume. The solu-
tion was stirred at 4 °C for 30 min and the final precipitate was pel-
leted by centrifugation (17,500g) at 4 °C for 10 min and then
resuspended in binding buffer I (0.5 M (NH4)2SO4, 50 mM Na2H-
PO4, 5% glycerol, 0.05% Triton X-100, pH 7.2). Recombinant GI-
GYF2(745–1030) was further purified by loading the sample onto
a HiTrap Phenyl HP column (1 mL, GE Healthcare UK Ltd, Bucking-
hamshire, UK) equilibrated in binding buffer I using an FPLC sys-
tem (GE Healthcare). The bound protein was subsequently eluted
with elution buffer I. Fractions containing GIGYF2(745–1030) were
pooled and loaded onto a HisTrap HP column (1 mL, GE Healthcare)
equilibrated in binding buffer II (7.8 mM Na2HPO4, 2.7 mM KCl,
1.5 mM KH2PO4, 0.5 M NaCl, 5% glycerol, 0.05% Triton X-100, pH
7.4) using an FPLC system. Finally, bound proteins were eluted
using elution buffer II (binding buffer II plus 0.5 M imidazole).
4.5. T7 phage display screen
Unless stated otherwise, all manipulations were performed at
room temperature. A 10 lM of Bio-RID-B solution was added to a
streptavidin-coated 96-well microplate (Nalge Nunc International,
Wiesbaden, Germany) and incubated for 1 h. The wells were then
blocked with 3% skimmed milk in TBS (25 mM Tris–HCl, 137 mM
NaCl, 2.7 mM KCl, pH 7.4) for 1 h. An aliquot of the T7 phage library
constructed from cDNA of Jurkat cells was added to each well and
the mixture was then incubated for 1 h. After incubation, the wells
were washed 10 times with TBST (TBS, 0.1% Tween 20). An elution
buffer (TBS, 1% SDS) was then added and the mixture incubated
for 15 min to recover the remaining phage particles. In order to
amplify the recovered phage particles, each eluate was mixed with
a culture of Esherishia coli BLT5615 (Merck) at a MOI of 0.001. The
cells were then cultured at 37 °C until cell lysis was observed. The
resulting solution was used for the next round of biopanning. After
four rounds of selection, the eluate was mixed with 1 mL of
BLT5615 culture and 10 mL of warmed top agarose and then
poured evenly across the surface of an LB/carbenicillin plate. Once
the overlay had solidified, the plate was incubated at 37 °C for 3 h
in order to allow the formation of phage plaques. A total of 15 pla-
ques were randomly picked and each plaque was suspended in
100 mM NaCl, 6 mM MgSO4, 20 mM Tris–HCl pH 8.0. The DNA se-
quence of each phage clone was then analyzed as described in a
previous report.16
4.9. Surface plasmon resonance assay
SPR analysis was performed on a BiacoreÒ 3000 (GE Health-
care). The purified GIGYF2(745–1030) was buffer exchanged into
PBS (67 mM Na2HPO4, 12.5 mM KH2PO4, 70 mM NaCl, pH 7.4)
and the concentration was quantified using DC protein assay kit
(Bio-Rad Laboratories, Hercules, CA). Solutions of bovine serum
albumin (BSA) at different concentrations were used as standards
for this assay. After immobilization of Bio-RID-B and biotin on an
SA sensor chip, appropriate concentrations of GIGYF2(745–1030)

S. Tsukuda et al. / Bioorg. Med. Chem. 21 (2013) 311–320
319
were injected over the flow cells. Binding analyses were carried out
in PBS at a flow rate of 20 L/min at 25 °C. The bulk effects of
DMSO were subtracted using reference flow cells. Kinetic parame-
ters were determined by analyzing the data using BIAevaluation
4.1 software (GE Healthcare).
then the lysates were placed on ice for 1 h. After centrifugation
(17,500g) at 4 °C for 30 min, the concentration of supernatant
was quantified using DC protein assay kit with BSA standards.
The supernatant was diluted to 2 mg/mL with 1% Triton lysis buffer
and mixed with an equal volume of 2ꢁ SDS sample buffer so that
the final protein concentration of the sample was 1 mg/mL. The
sample was then incubated at 96 °C for 10 min. The data was ana-
lyzed by Western blot using antibodies against Akt, phosphory-
lated Akt or GIGYF2.
l
4.10. Western blot analysis
Protein samples were separated by SDS–polyacrylamide gel
electrophoresis (SDS–PAGE) and transferred from the gel to a
polyvinylidene difluoride membrane (Merck) by electroblotting.
After blocking for 1 h, proteins were detected by Western blot
using primary antibody at the appropriate dilution and horserad-
ish peroxidase-conjugated anti-rabbit or anti-mouse IgG (GE
Healthcare) as the secondary antibody. The chemiluminescence
was performed using ECL western blotting detection reagents
(GE Healthcare) or ECL prime western blotting detection reagent
(GE Healthcare).
Acknowledgment
This study was partially supported by a Health Labour Sciences
Research Grant from the Ministry of Health Labour and Welfare,
Japan.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.037.
4.11. Cell culture and transfection
References and notes
HEK293T cells were maintained in DMEM (Nacalai Tesque) sup-
plemented with 10% fetal bovine serum (Life Technologies, Carls-
bad, CA) and 1% Penicillin–Streptomycin Mixed Solution (Nacalai
Tesque) at 37 °C under a humidified atmosphere of 5% CO2. Tran-
sient transfection was performed using the X-tremeGENE HP
DNA Transfection Reagent (Roche Diagnostics, Indianapolis, IN)
according to the manufacturer’s instructions.
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l
EDTA, 1% Triton X-100, protease inhibitor cocktail). The cell ly-
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then obtained by centrifugation (17,500g) at 4 °C for 30 min.
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0.1% Triton buffer with TBS. Avidin–agarose beads (Sigma–Al-
drich) were incubated with 10
lM of Bio-RID-B or DMSO and
then washed to remove unbound compounds. A 500
lL aliquot
of the diluted soluble fraction was then mixed with the avi-
din–agarose beads at 4 °C for 4 h. The beads were washed 6
times with wash buffer (0.1% Triton buffer in TBS) and the
bound proteins were subsequently eluted with 2ꢁ SDS sample
buffer (100 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 12% b-
mercaptoethanol, bromophenol blue). Samples were incubated
at 96 °C for 10 min and analyzed by Western blot using antibod-
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