Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction

Article abstract of DOI:10.1021/acsmedchemlett.8b00152

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3′ single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5′-exonu

Full text of DOI:10.1021/acsmedchemlett.8b00152

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Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric
Repeat Binding Factor 2 (TRF2) and Apollo Interaction
Xia Chen, Liu Liu, Yong Chen, Yuting Yang, Chao-Yie Yang,
Tianyue Guo, Ming Lei, Haiying Sun, and Shaomeng Wang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00152 • Publication Date (Web): 25 Apr 2018
Downloaded from http://pubs.acs.org on April 30, 2018
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ACS Medicinal Chemistry Letters
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Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric
Repeat Binding Factor 2 (TRF2) and Apollo Interaction
†
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┴
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║,£
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§
Xia Chen , Liu Liu , Yong Chen , Yuting Yang , ChaoꢀYie Yang , Tianyue Guo , Ming Lei , Haiying
†,
‡,
Sun * and Shaomeng Wang *
†
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China Pharmaceutical
University, Nanjing 210009, China
Department of Medicinal Chemistry, Department of Internal Medicine, Comprehensive Cancer Center, ┴Department of
Anesthesiology, University of Michigan, Ann Arbor, Michigan 48109, United States
State Key Laboratory of Molecular Biology, National Center for Protein Science, Shanghai Science Research Cenꢀ
‡
§
ter, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences; University of Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China
║
Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, 639 Zhizaoju Lu, Shanghai 200011, China
£
Shanghai Institute of Precision Medicine, Shanghai 200125, China
KEYWORDS: Telomere, TRF2, Apollo, Peptidic mimetics, Protein-Protein interaction
ABSTRACT: Telomeric repeat binding factor 2 (TRF2) is a telomereꢀassociated protein which plays an important role in the forꢀ
mation of the 3’ single strand DNA overhang and the “T loop”, two structures critical for the stability of the telomeres. Apollo is a
’ꢀexonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3’ single strand DNA overhang. Knocking
down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this paper we report
the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (ApolloTBM). We found conꢀ
formational control of the C terminal residues of ApolloTBM can effectively improve the binding affinity. We have obtained a crystal
structure of a cyclic peptidic Apollo peptide mimetic (34) complexed with TRF2 which provides valuable guidance to the future
design of TRF2 inhibitors.
5
Introduction
Shelterin is a telomere protecting complex consisting of
dues (amino acids 498ꢀ509), the so called TRFH binding motif
of Apollo (ApolloTBM), is clearly shown. Based on this strucꢀ
ture, Zhou et al. built a peptide library by replacing the amino
acids which have been shown in the crystal structure to interꢀ
act with the TRF2 protein with various natural amino acids.
They found that TRF2, through its TRFH domain can selecꢀ
six telomere proteins, including POT1, TPP1, TIN2, TRF1,
TRF2 and Rap1. Among these six components, TRF2 plays an
important role in the formation of the 3’ single strand DNA
overhang and the “T loop”, two structures critical for the staꢀ
22
1
ꢀ10
tively recognize peptides containing a [Y/F]XL sequence .
bility of the telomere . Knocking down TRF2 can cause endꢀ
toꢀend joining of DNA, induce DNA damage response, and
From this library they identified a consensus peptide which
binds potently to TRF2. Expression of this peptide in tandem
repeats in human cancer HTCꢀ75 cells promotes formation of
telomere dysfunctionꢀinduced foci (TIF) and induces DNA
damage response, suggesting that small molecular ligands
which can block the interactions between TRF2 and its associꢀ
ated proteins, such as Apollo, can modulate the functions of
TRF2.
11ꢀ13
eventually leads to cell senescence and apoptosis . Small
molecules which can interact with TRF2 and modulate its
functions are valuable tools in studies of shelterin and teloꢀ
meres. A series of stapled peptides which can potently bind to
TRF2 and block its interaction with Rap1 have been reported
14
recently , but small molecule inhibitors of TRF2 with differꢀ
ent mechanism of inhibition are still urgently needed.
In common with other peptides, Apollo and its analogues
have intrinsic drawbacks such as poor cell permeability and
poor stability, and consequently we have tried to design pepꢀ
tide mimetics of ApolloTBM to overcome these drawbacks
while maintaining or improving their binding affinity. In this
paper, we report our work on the design of ApolloTBM mimetꢀ
ics as inhibitors of TRF2.
TRF2 can recruit a number of accessory proteins whose
15, 16
functions are also critical for the protection of telomeres
.
One of these proteins is Apollo, a member of the mammalian
1
7, 18
SNM1/Pso2 family of nucleases
. Apollo contributes to the
formation of the singleꢀstranded 3’ telomere overhang, and its
deletion induces DNA damage response similar to that caused
19ꢀ21
by the knocking down of TRF2
. The crystal structure of an
Apollo peptide (residues 496ꢀ532) in a complex with the
TRFH domain of TRF2 has been determined. . In this strucꢀ
Results
10
ture (Figure 1) the electron density of the Nꢀterminal 12 resiꢀ
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To evaluate the binding affinity of our compounds to
Page 2 of 6
side chain of L500 in 5 binds to a large but shallow hydrophoꢀ
bic pocket in the TRF2 protein, and removal of this residue
decreases the binding by about 7 fold (6 vs 4). In the crystal
structure the methyl group of A501 can be seen to occupy a
small hydrophobic pocket in the TRF2 protein, while the side
chain of the neighboring residue, L502 is exposed to the solꢀ
vent and has no interaction with the protein. Our truncation
studies however indicate that removal of A501 doesn’t influꢀ
ence the binding affinity (7 vs 6), but removal of L502 deꢀ
creases the binding by about 5 fold (8 vs 7). These results sugꢀ
gest that these truncated peptides could bind to the TRF2 proꢀ
tein with a conformation different from that of the original
peptide 3. Compound 8 (KYLLTPV) binds only weakly to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TRF2, we designed a fluorescent tracer (compound 2, Figure
2) based on ApolloTBM and established a fluorescence polarizaꢀ
tion based binding assay. In the crystal structure of ApolloTBM
complexed with the TRFH domain of TRF2 (Figure 1), R498
of Apollo is exposed to the solvent and has no interaction with
the TRF2 protein. Consequently, in our design the R498 resiꢀ
due was replaced with a linker to which the fluorescence label
5
ꢀcarboxyꢀfluorescein (5ꢀFAM) is attached. Compound 2 has a
K value of 179 nM, consistent with the K value of Apollo
d
d
496ꢀ
10
in an ITC assay (K 120 nM) . The corresponding 11ꢀmer
5
32
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
peptide 3 (Table 1) has a K of 380 nM in our FP based bindꢀ
i
ing assay, thus proving the effectiveness of our assay.
TRF2 with a K of 122 ꢁM, so further truncation of this end of
i
the peptide was not pursued.
Table 1. Truncation studies for Apollo peptide
Compound Number
Peptides
GLALKYLLTPV
LALKYLLTPV
AcLALKYLLTPV
ALKYLLTPV
LKYLLTPV
Ki (µM)
0.38±0.12
2.2±0.34
0.34±0.08
22.8±2.6
26.8±3.2
122.6±8.4
1.89±0.23
26.3±2.7
98.0±9.6
461±34
3
4
5
6
7
8
9
KYLLTPV
AcLALKYLLTP
AcLALKYLLT
AcLALKYLL
AcLALKYL
1
0
11
1
2
Figure 1. Crystal structure of ApolloTBM in a complex with the
TRFH domain of TRF2 (PDB 3bua)
Cꢀterminal truncations were started from compound 5
which is as potent as 3. The crystal structure of ApolloTBM
complexed with TRF2 indicates that the Cꢀterminal residues of
the peptide have multiple hydrophobic interactions with the
protein. The side chain of V509 has a hydrophobic interaction
with M122 in TRF2, and we found that removal of this residue
decreases the binding affinity by a factor of about 5 fold (9 vs
HN
NH
2
OH
O
NH
O
O
O
O
H
H
N
H
N
H
N
H
N
H
N
N
N
NH
2
H
2
N
N
N
N
N
H
H
H
H
O
O
O
O
O
O
O
OH
NH
2
1 (ApolloTBM
)
5
). The pyrolidine ring of P508 has a hydrophobic interaction
OH
O
with Phe120 of TRF2 and Lei et al. found that mutation of
Phe120 to alanine completely abolishes the binding of TRF2
to Apollo, indicating that this hydrophobic interaction is critiꢀ
cal. We found that, consistent with the previous mutation
study, removal of P508 reduces the binding affinity by about
14 fold (10 vs 9). Notwithstanding the apparent absence of
interaction between T507 and the Apollo protein, removal of
T507 causes about a 4 fold decrease in binding affinity (11 vs
10), possibly because of conformational changes. The side
chain of L506 binds to a deep hydrophobic pocket in TRF2
and mutation studies performed by Lei et al. have confirmed
that this is the most important interaction in the binding of the
Apollo protein to TRF2. Our own truncation study supports
this in that after removal of L506 the resulting 5ꢀmer peptide
O
O
O
H
O
O
H
H
H
N
H
N
H
N
N
N
HO
N
N
H
N
NH
2
N
N
N
N
H
H
H
H
O
O
O
O
O
O
O
OH
O
COOH
NH
2
2
, K
d
179 ± 29.2 nM
O
Figure 2. Design of the fluorescent tracer for TRF2
We then performed truncation studies at both the Nꢀ
terminal and Cꢀterminal regions of the 11ꢀmer peptide (3) with
the purpose of identifying the shortest peptide sequence which
can maintain a reasonable binding affinity to Apollo, and the
results are summarized in Table 1. Of the Nꢀterminal truncaꢀ
tions, removal of the G499 residue decreases the binding by
about 7 fold (4 vs 3). In the crystal structure this residue has
no interaction with the protein, but its carbonyl group forms an
intramolecular hydrogen bond with the αꢀamino group of
K503 of Apollo. To assess the importance of this hydrogen
bond, we designed compound 5 in which the G499 residue in
(compound 12) retains only a very weak binding affinity to
TRF2 with a K of 461 ꢁM. Overall, our truncation studies
i
confirm the SAR information obtained from the ApolloTBM
ꢀ
TRF2 crystal structure, and indicate that among all the trunꢀ
cated peptides, only the acetylated 10ꢀmer peptide (5) mainꢀ
tains the good binding affinity. Consequently, this peptide was
used as the lead compound in further modifications.
3
is replaced by an acetyl group. With a K of 0.34 ꢁM, comꢀ
i
pound 5 is as potent as 3, indicating that the conformation
controlled by this hydrogen bond is critical to the binding. The
2
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Table 2. Structureꢀactivity relationship studies for compound 5.
1
2
3
4
5
6
7
8
9
Peptides
R1
R2
R3
R4
Binding affinity to
TRF2 (K , ꢀM)
i
1
1
1
3
4
5
methyl
3.2±0.40
3.4±0.52
0.72±0.13
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
methyl
propyl
>100
1
1
7
8
4.7±0.34
61.2±5.80
11.3±1.80
isopropyl
19
2
0
6.1±0.77
21
cyclopentyl
cyclohexyl
0.51±0.08
0.21±0.09
1.4±0.21
8.5±0.67
2
2
2
3
24
2
5
methyl
0.24±0.08
To gain additional structureꢀactivity relationships (SAR)
for this class of peptides, we performed mutation studies for
several residues which have been shown to be critical to the
binding affinity and the results are reported in Table 2. The
ApolloTBMꢀTRF2 crystal structure indicates that the εꢀamino
group of K503 and the hydroxyl group of Y504 of Apollo
have electrostatic interactions with Glu94 of TRF2 (Figure 1).
In order to explore the significance of these interactions, we
designed three mutated peptides 13ꢁ15. In 13, K503 was reꢀ
placed with an alanine, while in 14 and 15, the side chain of
Y504 was replaced with benzyl or a 4ꢀaminobenzyl, respecꢀ
tively. 13 and 14 are about 10 times less potent than 5 with Ki
values of 3.2 and 3.4 µM respectively, suggesting that the
electrostatic interactions from both K503 and Y504 are imꢀ
an alanine and found the resulting compound (25) is as potent
as 5, therefore subsequently, 25 was used as the new lead
compound for further modifications.
Figure 3. Design of cyclic Apollo peptide mimetics
From the crystal structure, we observed that the six Nꢀ
terminal residues GLALKY in 3 are part of an αꢀhelix, while
the five Cꢀterminal residues LLTPV form a flexible loop
(Figure 1). In αꢀhelix, the peptide bonds are all involved in
intramolecular hydrogen bonds, with only the side chains parꢀ
ticipating in the interactions with the proteins, so for Nꢀ
terminal residues we planned to replace the helical peptide
with a nonꢀpeptide scaffold to which different substituent
groups could be attached to mimic the side chains of the resiꢀ
dues which interact with the protein. For the Cꢀterminal resiꢀ
dues, the conformational flexibility is a disadvantage for modꢀ
ification because it can compromise the SAR, and thus we
sought to constrain the conformation flexibility of these resiꢀ
dues. In the crystal structure the side chains of L505 and V509
are very close to one another (Figure 1). Therefore, we proꢀ
posed that these two residues could be linked to form a conꢀ
formationally constrained cyclic peptide.
portant to the binding affinity. 15, with a K of 0.72 µM, is
i
only 2 times less potent than 5 and about 5 times more potent
than 14, confirming the importance of the electrostatic interacꢀ
tion between 15 and the Glu94 residue in the protein. Because
the hydrophobic interaction of the side chain of L506 with a
deep hydrophobic pocket in TRF2 has been shown to be the
most critical to the binding affinity, we performed extensive
modifications to this residue, replacing it with a series of natuꢀ
ral and unnatural amino acids containing different hydrophoꢀ
bic groups. Among all the amino acids tested, the binding afꢀ
finity is maintained or slightly improved only when L506 is
replaced with cyclopentyl glycine (21) or cyclohexyl glycine
(
22). All other modifications at this site result in a decrease in
the binding affinity. T507 has no interaction with the protein,
but the hydroxyl group in this threonine residue can cause
some synthetic inconvenience due to the need for protection
and deprotection. Accordingly, we replaced this residue with
To test this strategy, we first designed and synthesized a
cyclic peptide 27 (Figure 3) by replacing the side chains of
3
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O
O
O
N
N
N
O
N
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
O
N
H
H
H
O
NH
O
O
NH
O
O
O
O
NH
O
5
O
O
NH
NH
NH
N
N
N
N
N
N
H
(CH
2
)
5
H
NH
2
2 4
(CH )
NH
2
H
(
CH
2
)
NH
2
O
O
HO
O
2
8, K
i
58.2 ± 4.9 µM
29, K
i
41.4 ± 3.1 µM
30, K
i
130.3 ± 8.9 µM
NH
2
NH
2
O
O
O
N
N
N
N
N
O
N
H
H
H
O
NH
O
O
O
NH
O
O
O
NH
O
O
O
O
O
NH
H
O
NH
H
H
NH
N
AcHN
N
N
N
N
H
2
N
N
N
N
N
N
N
(CH
2
)
6
H
(CH
2
)
5
H
H
H
(CH2)5
H
NH
2
O
O
O
O
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31, K
i
43.2 ± 2.7 µM
32, K
i
18.4 ± 2.1 µM
33, K 0.09 ± 0.02 µM
i
OH
OH
O
O
N
O
N
N
N
H
H
O
NH
O
O
O
NH
O
H
O
H
N
NH
N
O
NH
H
2
N
N
N
N
N
O
(CH )
O
(CH2)5
H
2 5
H
O
O
34, K
i
14.3 ± 1.9 µM
35, K
i
11.2 ± 1.6 µM
Figure 4. Structures and activities of cyclic peptide mimetics of Apollo peptides
L505 and V509 in the Cꢀterminal 5ꢀmer Apollo peptide (26)
with a linker containing seven methylene groups correspondꢀ
ing to the distance between these two side chains in the crystal
structure. In our FP based binding assay, compound 27 with a
K of 115 ꢁM, is at least three times more potent than the 5ꢀ
mer peptide 26 (K > 500 ꢁM).
five residues of ApolloTBM. The conformation of the protein is
very close to that of ApolloTBM with TRF2, with the exception
of the helix containing Glu94. When binding to the wild type
peptide, this helix is distorted because of the interactions of
Glu94 with the εꢀamino group in K503 and the OH group in
Y504 (Figure 1). But when binding to 34, Glu94 flips back
and has no interaction with the compound, suggesting that the
interaction between Glu94 and the amino group in 34 is not
sufficient to cause the distortion of the helix. This also exꢀ
plains why the Tyrꢀcontaining compound 28 is as potent as the
Pheꢀcontaining compound 29. The amino group of (S)ꢀ3ꢀ
aminoꢀ4ꢀphenylbutyric acid is exposed to the solvent, while its
phenyl group binds to a large hydrophobic area where the Nꢀ
terminal residues bind.
i
i
Encouraged with the improved binding affinity for 27 over
2
6, we then designed compounds 28 and 29 by introducing a
Tyr or Phe to the amino group of 27 (Figure 4). Compounds
8 and 29 were found to have comparable binding affinity to
2
TRF2 with K values of 58 and 41 ꢁM respectively, and are 3
i
to 4 times more potent than 27, suggesting that the hydroxyl
group in 28 may fail to interact with Glu94 in the TRF2 proꢀ
tein. We have explored the influence of the length of linker to
the binding affinity of the cyclic peptides by designing comꢀ
pounds 30 and 31 (Figure 3) by replacing the 7 methylene
linker in 29 with a spacer containing 6 or 8 methylene groups,
respectively. The peptide 30 is 3 times less potent than 29 but
3
1 is as potent as 29, indicating that linkers with 7 or 8 methꢀ
ylene groups are more optimal for cyclic peptides. In order to
confirm that a cyclic peptide can be used to replace the Cꢀ
terminal linker peptide, we designed the 7ꢀmer analogue 32
and the 10ꢀmer analogue 33 by reꢀintroduction of the approꢀ
priate ApolloTBM Nꢀterminal residues to 28. Compound 32
binds to TRF2 with a K of 18 ꢁM, and is 7 times more potent
than the 7ꢀmer peptide 8, while 33 binds to TRF2 with a K of
0 nM, and is 3 times more potent than the 10ꢀmer peptide 5,
proving that conformational control of the Cꢀterminal residues
i
i
9
can effectively improve the binding affinity.
We also examined Nꢀterminal modifications of 29. Comꢀ
pound 34, in which a (S)ꢀ3ꢀaminoꢀ4ꢀphenylbutyric acid was
introduced to the free amino group of 29, has a K of 14.7 ꢁM,
and is about 3 times more potent than 29. The synthetic cyclic
peptidic mimetics have been screened by coꢀcrystallization
with the TRF2 TRFH domain protein. The complex of 34 with
TRF2 gave a crystal structure with a resolution of 2.05 Å
i
Figure 5. Crystal structure of compound 34 in a complex with
TRF2
Based on this crystal structure, we designed compound 35
(Figure 4) by introduction of a 3ꢀphenylpropanoic acid to the
amino group of 29. This compound has a Ki of 11.2 µM, indiꢀ
(
Figure 5). In this crystal structure, 34 can be seen to bind to
the same area in the TRFH domain of TRF2 as the Cꢀterminal
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cating that introduction of a hydrophobic group to the Nꢀ
terminal indeed improves the binding affinity. This crystal
structure thus provides the foundation for our future design.
(1) Cristofari, G.; Sikora, K.; Lingner, J. Telomerase Unplugged. ACS
Chem. Biol. 2007, 2, 155ꢀ158.
2) de Lange, T. Shelterin: the protein complex that shapes and safeguards
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human telomeres. Genes Dev. 2005, 19, 2100ꢀ2110.
Summary
(3) Court, R.; Chapman, L.; Fairall, L.; Rhodes, D. How the human
telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view
from high resolution crystal structures. EMBO Rep. 2005, 6, 39ꢀ45.
(4) O'Connor, M. S.; Safari, A.; Xin, H.; Liu, D.; Songyang, Z. A critical
role for TPP1 and TIN2 interaction in highꢀorder telomeric complex
assembly. Proc. Acad. Sci. U. S. A. 2006, 103, 11874ꢀ11879.
We have designed a fluorescent tracer based on the crystal
structure of ApolloTBM in a complex with the TRFH domain of
TRF2 and developed a competitive binding assay based on
fluorescenceꢀpolarization for this domain of TRF2. Through
truncation and mutation studies we identified an acetylated 10ꢀ
mer peptide (25) which can bind to TRF2 with a binding affinꢀ
ity comparable to that of ApolloTBM. Based on the Cꢀterminal
five residues in 25, we designed and synthesized a series of
cyclopeptidic mimetics and found that replacement of the five
Cꢀterminal residues in 25 with our designed cyclic peptide can
effectively improve the binding affinity. Our study has yielded
(5) Lei, M.; Podell, E. R.; Cech, T. R. Structure of human POT1 bound to
telomeric singleꢀstranded DNA provides a model for chromosome endꢀ
protection. Nat. Struct. Mol. Biol. 2004, 11, 1223.
(6) Baumann, P.; Cech, T. R. Pot1, the Putative Telomere EndꢀBinding
Protein in Fission Yeast and Humans. Science 2001, 292, 1171ꢀ1175.
(7) Loayza, D.; de Lange, T. POT1 as a terminal transducer of TRF1
telomere length control. Nature 2003, 423, 1013.
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(8) de Lange, T. Tꢀloops and the origin of telomeres. Nat. Rev. Mol. Cell
Biol. 2004, 5, 323.
a cyclic peptide 33, which binds to TRF2 with a K value of 90
i
(9) Smogorzewska, A.; Lange, T. d. Regulation of Telomerase by
nM. We solved the crystal structure of one of our cyclic pepꢀ
tide analogues (34) in a complex with TRF2, and found that
this compound binds to the same site in TRF2 as the original
ApolloTBM peptide. Further optimizations to these cyclic pepꢀ
tides are in progress and the results will be reported subseꢀ
quently.
Telomeric Proteins. Annu. Rev. Biochem. 2004, 73, 177ꢀ208.
(10) Chen, Y.; Yang, Y.; van Overbeek, M.; Donigian, J. R.; Baciu, P.; de
Lange, T.; Lei, M. A Shared Docking Motif in TRF1 and TRF2 Used for
Differential Recruitment of Telomeric Proteins. Science 2008, 319, 1092ꢀ
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(11) Zhang, Y.ꢀW.; Zhang, Z.ꢀX.; Miao, Z.ꢀH.; Ding, J. The Telomeric
Protein TRF2 Is Critical for the Protection of A549 Cells from Both
Telomere Erosion and DNA DoubleꢀStrand Breaks Driven by Salvicine.
Mol. Pharm. 2008, 73, 824ꢀ832.
ASSOCIATED CONTENT
Supporting Information
(12) Grolimund, L.; Aeby, E.; Hamelin, R.; Armand, F.; Chiappe, D.;
Moniatte, M.; Lingner, J. A quantitative telomeric chromatin isolation
protocol identifies different telomeric states. Nat. Commun. 2013, 4, 2848.
The procedure and experimental detail for solid phase synthesis of
the truncated and mutated peptides, chemical synthesis of the
cyclic peptidic mimetics, saturation binding experiment to deterꢀ
(13) Stagno D'Alcontres, M.; MendezꢀBermudez, A.; Foxon, J. L.; Royle,
N. J.; Salomoni, P. Lack of TRF2 in ALT cells causes PMLꢀdependent
p53 activation and loss of telomeric DNA. J. Cell Biol. 2007, 179, 855ꢀ
867.
mine dissociation constant (K ) of the tracer, competitive binding
d
experiment to determine the binding affinities of the synthesized
compounds, crystallization of the complex of 34 with TRF2 and
structural determination of the crystal
(14) Ran, X.; Liu, L.; Yang, C.ꢀY.; Lu, J.; Chen, Y.; Lei, M.; Wang, S.
Design of HighꢀAffinity Stapled Peptides To Target the Repressor
Activator Protein 1 (RAP1)/Telomeric RepeatꢀBinding Factor 2 (TRF2)
Protein–Protein Interaction in the Shelterin Complex. J. Med. Chem. 2016,
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9, 328ꢀ334.
15) de Lange, T. Telomeres and Senescence: Ending the Debate. Science
998, 279, 334ꢀ335.
The Supporting Information is available free of charge on the
ACS Publications website.
(16) van Steensel, B.; Smogorzewska, A.; de Lange, T. TRF2 Protects
Human Telomeres from EndꢀtoꢀEnd Fusions. Cell 1998, 92, 401ꢀ413.
AUTHOR INFORMATION
Corresponding Author
(17) van Overbeek, M.; de Lange, T. Apollo, an ArtemisꢀRelated
Nuclease, Interacts with TRF2 and Protects Human Telomeres in S Phase.
Curr. Biol. 2006, 16, 1295ꢀ1302.
* Eꢀmail: haiyings1969@gmail.com
* Eꢀmail: shaomeng@med.umich.edu
(18) Lenain, C.; Bauwens, S.; Amiard, S.; Brunori, M.; GiraudꢀPanis, M.ꢀ
J.; Gilson, E. The Apollo 5' Exonuclease Functions Together with TRF2 to
Protect Telomeres from DNA Repair. Curr. Biol. 2006, 16, 1303ꢀ1310.
Author Contributions
(19) Akhter, S.; Lam, Y. C.; Chang, S.; Legerski, R. J. The telomeric
All authors have given approval to the final version of the manuꢀ
script.
protein SNM1B/Apollo is required for normal cell proliferation and
embryonic development. Aging Cell 2010, 9, 1047ꢀ1056.
(20) Liu, L.; Akhter, S.; Bae, J.ꢀB.; Mukhopadhyay, S.; Richie, C.; Liu,
Funding Sources
X.; Legerski, R. SNM1B/Apollo interacts with Astrin and is required for
the prophase cell cycle checkpoint. Cell Cycle 2009; 8, 628ꢀ638.
(21) Demuth, I.; S Bradshaw, P.; Lindner, A.; Anders, M.; Heinrich, S.;
Kallenbach, J.; Schmelz, K.; Digweed, M.; Stephen Meyn, M.;
Concannon, P. Endogenous hSNM1B/Apollo interacts with TRF2 and
stimulates ATM in response to ionizing radiation. DNA Repair 2008; 7,
Financial support is from national natural science foundation of
China 81473079, the Program for Jiangsu Province Innovative
Research Team, the Program for Jiangsu Province Innovative
Talent and the Program for Specially Appointed Professor of
Jiangsu Province to Haiying Sun.
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192ꢀ1201.
Notes
(22) Kim, H.; Lee, O.ꢀH.; Xin, H.; Chen, L.ꢀY.; Qin, J.; Chae, H. K.; Lin,
The authors declare no competing financial interest.
S.ꢀY.; Safari, A.; Liu, D.; Songyang, Z. TRF2 functions as a protein hub
and regulates telomere maintenance by recognizing specific peptide
motifs. Nat. Struct. Mol. Biol. 2009, 16, 372ꢀ379.
ACKNOWLEDGMENT
The authors thank Dr. Bill Milne for the proof reading of the
manuscript.
REFERENCES
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