Design and synthesis of fluorescent and biotin tagged probes for the study of molecular actions of FAF1 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2016.01.045

To study the molecular action of ischemic Fas-mediated cell death inhibitor, we prepared fluorescent-tagged and biotin-tagged probes of the potent inhibitor, KR-33494, of ischemic cell death. We used the molecular modeling technique to find the proper position for attaching those probes with minimum interference in the binding process of probes with Fas-mediated cell death target, FAF1.

Full text of DOI:10.1016/j.bmcl.2016.01.045

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1169–1172
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of fluorescent and biotin tagged probes for the
study of molecular actions of FAF1 inhibitor
a,
⇑
b
a
b
a,
⇑
Sung-eun Yoo , Changsun Yu , SeoHee Jung , Eunhee Kim , Nam Sook Kang
a
Graduate School of New Drug Discovery and Development, Chungnam National University, Daehakno 99, Yuseong-gu, Daejeon 305-764, Republic of Korea
Department of Bioscience and Biotechnology, Chungnam National University, Daehakno 99, Yuseong-gu, Daejeon 305-764, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
To study the molecular action of ischemic Fas-mediated cell death inhibitor, we prepared fluorescent-
tagged and biotin-tagged probes of the potent inhibitor, KR-33494, of ischemic cell death. We used the
molecular modeling technique to find the proper position for attaching those probes with minimum
interference in the binding process of probes with Fas-mediated cell death target, FAF1.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 30 September 2015
Revised 12 January 2016
Accepted 16 January 2016
Available online 18 January 2016
Keywords:
Fas-mediated cell death pathways
FAF1
Biotin tagged probe
Fluorescence tagged probe
KR-33493
Ischemia is caused by contraction or occlusion of the blood ves-
sels. Although the mechanisms of cell death after ischemic attack
are not fully understood yet, Fas and FasL are known to be impor-
In order to study the molecular action of FAF1 inhibitor, KR-
33493, in the ischemic condition, we need to prepare fluores-
cent-tagged and biotin-tagged probes. First, we needed to find
1
tant factors in the pathology of ischemia together with FAF1 pro-
2
–4
tein.
Through the extensive designing and derivatizing process
we have identified KR-33493 (Fig. 1) as a potent inhibitor for
Fas-mediated cell death, FAF1.5
,6
Figure 2. Molecular model for the complex of FAF1 (green) and KR-33493
compound (orange), which is obtained through the hierarchical protein structure
modeling approach, based on secondary-structure enhanced Profile–Profile thread-
ing Alignment (PPA) and the iterative implementation of the Threading ASSEmbly
Figure 1. The structure of KR-33493.
Refinement (TASSER) program. The Phenyl rings of Tyr2 and Trp
25
240
play an
⇑
Corresponding authors.
important role in interaction with CIF forming p–p stacking interaction.
http://dx.doi.org/10.1016/j.bmcl.2016.01.045
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0

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S.-e. Yoo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1169–1172
interaction of KR-33493 with FAF1 protein. To find the binding
mode of KR-33493 with FAF1, we initially constructed the 3D
structure of FAF1 and then carried out the docking experiments
as shown in Figures 2 and 3. As the sequence similarity of FAF1
with other proteins is less than 20%, we used hierarchical protein
structure modeling approach based on the secondary-structure
enhanced Profile–Profile threading Alignment (PPA) and the itera-
tive implementation of the Threading ASSEmbly Refinement (TAS-
7
,8
SER) program. Through this program, we obtained five candidate
models for 3D structure of FAF1 and thus carried out molecular
dynamics simulation using CHARMM force field (version 27.0)
and default parameters interfaced with Accelrys Discovery Stu-
dio3.5. To decide binding sites, we used the binding site defining
protocol in Accelrys Discovery Studio3.5 and defined the binding
site from receptor cavity. Then, we docked our compounds includ-
ing KR-33493 and an inactive compounds from our previous
9
study, and thus examined whether or not match SAR study for
the compounds, and finally selected one binding model for FAF1
and an active compound as shown in Figure 1. In this binding mode
the carbonyl group of Ser229 acts as a hydrogen bonding acceptor
to form a hydrogen-bonding with the amide group of KR-33493
and the hydroxyl group on the side chain of Ser229 forms another
hydrogen bonding with the carboxyl group of KR-33493. The
amine group of Met323 acts as a hydrogen-bonding donor to form
hydrogen bonding with the amide carbonyl group of KR-33493.
Especially, Met323 together with Met322 seems to provide a
rather narrow hydrophobic environment.
Figure 3. One of possible models for binding interaction of KR-33493 with FAF1.
the position for the attachment of fluorescent and biotin
components. The most preferred position to attach these compo-
nents would be the one that has a least interference in the binding
O
N
O
N
O
2
N
O
O N
2
O
O
N
O
N
O
2
N
OH
O
2
N
O
N
N
N
H
N
H
(A)
(B)
(C)
O
OH
O
N
H
O
O
(
D)
Br
Br
O
N
O
N
O
O
N
S
S
Br
NH
O
NH
O
NH
O
2
H N
O
O
O
N
N
N
N
N
(G)
(F)
(E)
O
O
O
O
I
O
O
O
O
NH
2
N
H
N
H
O
N
H
O
Br
(H)
(I)
Br
O
N
S
O
N
S
NH
OH
NH
OH
O
O
N
N
N
O
HN
H
O
O
O
NH
N
O
S
N
H
H
H
O
S
III
II
Scheme 1. Reagents and condition: (A) concd sulfuric acid, MeOH, rt, 92%; (B) 4-(2-bromo-ethyl)-phenol, Cs
rt, 97%; (D) 10% Pd/C, H , MeOH, rt, 97%; (E), (F) bromoacetyl bromide, p-bromobenzenethiol, NEt , THF, rt, 76%; (G) trifluoroacetic acid, CH
succinimide, EDCÁHCl, DMAP, NEt , DMF, rt (ii) 6 N NaOH, MeOH, rt, 73%; (I) (i) dansyl chloride, NEt , CH Cl , 0 °C, (ii) 6 N NaOH, MeOH, rt, 84%.
2
CO
3
, DMF, rt, 56%; (C) t-boc aminobutyl bromide, Cs
2 3
CO , DMF,
2
3
2
Cl , 0 °C, 89%; (H) (i) D-biotin, N-
2
3
3
2
2

S.-e. Yoo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1169–1172
1171
This analysis suggests that the para position of the phenethyl
part of KR-33493 would be a good position for the attachment of
biotin and fluorescent components. For the convenience reason,
we decided to prepare a common linker with a proper length hav-
ing an amine functional group as in the intermediate I in Scheme 1.
We then perform the computer modeling study on the biotin
attached compound II and dansyl, 5-(dimethylamino)naph-
thalene-1-sulfonyl, group attached compound III to examine
whether these compounds fit well in our model without interfer-
ence in the binding interaction of KR-33493 and FAF1. The model-
ing study indicates that these probes sit well in the narrow groove
without significant interactions with neighboring amino acids as
shown in Figure 4. The synthesis and the reaction condition for
the intermediate I and the compounds II and III are shown in
Scheme 1.
biotin- or dansyl-tagged KR-33493 retain the protective potential
of parental compound KR-33493.
Also, to examine whether FAF1 is the cellular target of KR-
33493 using compound II, the SH-SY5Y cells were transfected with
2 2
the Flag-FAF1. After 36 h, cells were treated with H O 500 lM for
30 min and then lysed in lysis buffer containing 50 mM Tris–HCl
(pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 and
0.4 mM phenylmethylsulfonylfluoride. Biotinylated KR-33493
First of all, to validate whether the biotin- or dansyl-tagged KR-
3
3493 retain the activity of the parent compound KR-33493, we
examined its ability to block the cell death. The SH-SY5Y cells were
seeded into 96-well plates and incubated for 24 h. The SH-SY5Y
cells then were pretreated with dimethyl sulfoxide (DMSO; vehi-
cle), KR-33493 (20
tagged KR-33493 (20
were treated with 500
KR-33493, biotin-tagged KR-33493 or dansyl-tagged KR-33493.
Cell death was assessed by the release of lactate dehydrogenase
l
M), biotin-tagged KR-33493 (20
M) for 30 min. After pretreatment, cells
M H for 6 h in presence of vehicle,
lM) or dansyl
l
Figure 6. SH-SY5Y cells were transfected with Flag-FAF1. After 36 h, cells were
l
2 2
O
treated with H
prepared from SH-SY5Y cells exposed to H
bound agarose or biotinylated KR-33493 (100
2
O
2
500
l
M for 30 min and then lysed in lysis buffer. The cell lysates
were incubated with either biotin-
M)-bound agarose for 12 h at 4 °C.
2 2
O
l
(
LDH) into the extracellular medium. Cell viability was determined
The agarose beads were then washed five times with lysis buffer and were
subjected to immunoblot analysis with anti-Flag.
by MTS assay. As shown in Figure 5, these results demonstrate that
Figure 4. (A) The induced-fit docking model of Biotin-KR-33493 (compound II) (B) Dansyl tagged compound (compound III). Images were made using Accelrys Discovery
studio4.0.
Figure 5. SH-SY5Y cells were pretreated with vehicle (DMSO), KR-33493 (20
were treated with 500 M H for 6 h in presence of vehicle or individual compounds. (A) Cell death was determined by measuring LDH release. (B) Cell viability was
determined by MTS assay.
lM), biotin-tagged KR-33493 (20 lM) or dansyl-tagged KR-33493 (20 lM) for 30 min. Cells then
l
2 2
O

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S.-e. Yoo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1169–1172
5
4
4
3
3
2
2
1
1
00
50
00
50
00
50
00
50
00
of non-treated cells as shown in Figure 6. This result indicated that
KR-33493 targets FAF1 in a stimulus-dependent manner. Also, the
dansyl group attached probe, III, shows high intensity spectrum
with the excitation max. at 325 nM and emission max. at 468 nM
as shown in Figure 7.
In this study, to study the molecular action of ischemic Fas-
mediated cell death inhibitor, we prepared fluorescent-tagged
and biotin-tagged probes of the potent inhibitor, KR-33494, of
ischemic cell death and we confirmed FAF1is the cellular target
of KR-33493 using the SH-SY5Y cells transfected with the Flag-
FAF1. Afterward, we will try to use these kinds of probes to identify
the biological mode of action for some molecules.
5
0
0
Acknowledgment
3
00
350
400
450
500
550
600
This study was financially supported by research fund of
Chungnam National University, South Korea, in 2013.
Wavelength (nM)
Figure 7. The Fluorescence Excitation Spectrum in green color (max. intensity at
25 nM) and the Dispersed Fluorescence Spectrum in brown color (max. intensity
at 468 nM) taken by Perkin Elmer LS55.
3
References and notes
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was preincubated with 7
4
3
for 12 h at 4 °C. The beads were washed five times with lysis buffer,
and compound-bound proteins were subjected to immunoblot
analysis with anti-Flag. When the biotinylated KR-33493 (II) was
incubated with cell lysates prepared from the Flag-FAF1 expressing
SY-SY5Y cells exposed to H
conjugated agarose beads, strong interaction between compound II
and FAF1 was detected in lysates of H -treated cells than those
l
l streptavidin agarose (Sigma) for 4 h at
°C. The beads were then isolated by centrifugation. The KR-
3493-bound agrose beads were then incubated with cell lysates
3
4
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7
8
9
.
.
.
2 2
O and pulled down with streptavidin-
2 2
O