Switching substitution groups on the in-tether chiral centre influences backbone peptides' permeability and target binding affinity

Article abstract of DOI:10.1039/c6ob02289h

Different substitution groups on the in-tether chiral centre of chirality-induced helical peptides (CIH peptides) showed distinguishable effects on the peptides' cellular uptakes and binding affinities with the estrogen receptor α(ER-α). This study proves that in-tether chiral centres are a valuable modification site for constructing peptide ligands with preferable biophysical properties.

Full text of DOI:10.1039/c6ob02289h

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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Org aP nl ei ac s &e dB oi on mo to al ed cj uu sl at rm Ca hr ge imn si stry
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DOI: 10.1039/C6OB02289H
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Switching Substitution Groups on the In-tether Chiral Centre
Influences the Backbone Peptide’ Permeability and Target Binding
Affinity
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
c
d
a
DOI: 10.1039/x0xx00000x
Yixiang Jiang, Kuan Hu, Xiaodong Shi, Qingzhuang Tang, ZiChen Wang, Xiyang Ye* , Zigang Li*
www.rsc.org/
Different substitution groups on the in-tether chiral centre of the
chirality-induced helical peptides (CIH peptides) showed
distinguishable effects on peptides’ cellular uptakes and binding
affinities with estrogen receptor α(ER-α). This study proves that
in-tether chiral centre is a valuable modification site for
constructing peptide ligands with preferable biophysical
properties.
R
S
S
i+4
i+4
i+4
i
i
i
A
B
Moore's work
Our previous work
This work
C
Protein-protein interactions (PPIs) play pivotal roles in
mediating intracellular biological processes and targeting
dysfunctional 1 PPIs is broadly utilized for therapeutics
development. However, small molecule ligands are less likely
to interrupt PPIs with large, shallow or discontinued surfaces,
2
Fig. 1 (A)Stabilized peptides with an in-tether chiral centre developed by Moore
et al. and us. (B) Crystal structure of stapled peptide bound to estrogen receptor.
which makes many PPIs “undruggable”. Over 50% PPIs involve
α-helices interactions, however; although peptides could
efficiently interrupt PPIs in vitro, they suffer from poor stability
and cell permeability. In past decade or so, constraint helical
peptides stabilized by various chemical means with enhance
druggability were intensively studied to construct suitable
Although conceptually the artificial tethers were designed
to point at the solvent face to avoid direct interactions with
target protein; however, in some reported cases, the tether
counted significantly for the overall ligand-target interaction,
such as ER-α and MDM-2. Phillips et al. reported an example of
replacing interacting residues with a hydrocarbon staple. The
crystal structure of stapled peptide PFE-SP2 bound to estrogen
3
peptide-ligands for various PPIs.
Recently, Moore et al. reported that a chiral centre on the
tether of a stapled peptide could affect the peptide’s
secondary structure and binding affinity. Meanwhile, we
reported that a precisely positioned carbon chiral centre in a
single bonded tether was capable of modulating the helicity,
receptor
α (ERα) showed that an i, i + 4 hydrocarbon staple
can replace isoleucine and leucine residues on the binding face
of a steroid receptor coactivator 2 (SRC2) peptide (Fig. 1B).
They found that this change receives an increase in helical
content and binding affinity. SRC2 interacts with the surface of
ERa over two turns of an a-helix using an LXXLL motif (X is any
4
cell permeability and binding affinity of a peptide. Both
groups clearly stated that the substitution group on the in-
tether chiral centre could be of important modulating effects
on backbone peptides’ biophysical properties, without any
alteration on the backbone peptides (Fig. 1A).
5
amino acid). Moore et al. also reported the complex structure
of the chiral center bearing stapled peptide with ERα in a
4
similar pattern (Fig. 1C). . In Moore’s and our previous reports,
both of us found the in-tether chiral centre showed significan4t
influences on the peptides’ binding affinity with ER-α.
However, how the substitution groups on the in-tether chiral
centre influence the backbone peptides’ biophysical properties
still lacks systematic study.
In this report, a panel of CIH peptides targeting ER-α with
identical peptide backbone but different branches at the in-
tether chiral centre were constructed as shown in Fig. 2A. Six
a.
School of Chemical Biology and Biotechnology, Peking University Shenzhen
Graduate School, Shenzhen, 518055, China. E-mail: lizg@pkusz.edu.cn
Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong
Kong SAR, China.
Shenzhen Middle School, Shenzhen 518001, China.
Department of Gynecology, Shenzhen People’s hospital, Shenzhen 518055, China.
b.
c.
d.
Electronic Supplementary Information (ESI) available: experimental procedures,
supporting tables and figures. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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unnatural amino acids with different branches utilized in this
Journal Name
The fluorescein-labelled CIH peptides FITC-1a/1b to 6
View Article Online
6
DOI: 10.1039/C6OB02289H
research were shown in Fig. 2B. Since Moore et al. reported in were tested for their binding affinity with ER-α by fluorescence
their study that the stapled peptide without a chiral centre polarization (FP) assay (Fig. 4A). Peptide
showed better helical content but minimal binding with ER-α significantly better binding with ER-α comparing with their
comparing with stapled peptides containing a chiral centre, counterparts and selected examples were shown in Fig. 4B.
b epimers showed
a
therefore, we prepared peptide
chiral centre as a control.
6
, which contains no in-tether Binding curves of other peptides were summarized in Fig. S2
and Table S2. The binding affinity of peptide FITC-1b to was
6
summarized in Fig. 4D. From FITC-1b to FITC-5b, the binding
affinity correlated well with the peptide’s helicity as shown in
Fig. 4C, which clearly indicated that helicity is an important
factor for CIH peptides’ binding affinity. However, as an
R
SH
A
S
i
3
65 nm
i
i+4
R
2
h, DMF
i+4
Photo-induced intramolecular thiol-ene coupling
outliner, peptide
6 showed both the best helicity and the
R= H, Me, Et, iPr, Ph, Bn
poorest binding affinity (Fig. 4C/4D). This observation clearly
demonstrated that the substitution groups may contribute
significantly for the peptide/target interaction in addition to
maintaining the helical structure, which is in agreement with
B
4
Moore et al.’s previous report. Peptide 1b exhibited the best
C
binding affinity, which may be attributed to appropriate size
and hydrophobicity of the substitution group.
1a
1b
1linear
A
B
D
C
Fig. 2 (A) Schematic presentation of CIH peptide preparation. (B) Structures of
unnatural amino acids used in this study. S (2-Me) means there is a methyl group
at the γ-position to the S amino acid. (C) HPLC traces of epimers 1a, 1b and
5
1
linear. (D) Peptide sequences.
Fig. 3 (A) CD spectra of linear peptide 1 and 1a/1b in 20% TFE 10 mM phosphate
buffer solution, pH=7.4, at 20°C. (B) CD spectra of b epimers of all different chiral
Peptides with different substitution groups were
synthesized and the epimers were readily separated by centres and H atom as a control in 20% TFE 10 mM phosphate buffer solution,
reverse-phase HPLC. The retention time differences clearly
indicated significant structural differences in solution as shown
pH=7.4, at 20°C. (C) Percentage of helicity of all b epimers, each peptide was
7
calculated as previous report .
in Fig. 2C. Circular dichroism(CD) spectroscopy analysis was
A
performed on linear peptide
obtained by cyclization of . Peptide 1b showed significantly
enhanced helical contents than its epimer 1a while the linear
peptide was mainly random coil. (Fig. 3A) The CD spectra of
all epimers were summarized in Fig. 3B and the α-helical
content of each b epimer was calculated as previous reports
Fig. 3C). CD spectra of epimers were summarized in
Supporting Information Fig. S1 and Table S1. Notably, we also
detected peptide exhibited better helical content comparing
1, and two epimers 1a and 1b
1
1
b
7
B
D
C
(
a
6
with other CIH4 peptides, which was in agreement with
Moore’s report. Remarkably, it is the only case identified in
our laboratory that a control cyclic peptide showed better
helical enhancement than CIH peptides.
Peptide
FITC-1b
FITC-2b
FITC-3b
FITC-4b
FITC-5b
FITC-6
Sequence
FITC-βA-R[cyclo-CILHS (2-Me)]LLQDS-NH
FITC-βA-R[cyclo-CILHS
Kd for ER-α（nM）
5
2
2
42
5
(2-Et)]LLQDS-NH
5
(2-iPr)]LLQDS-NH
2
167
196
75
252
415
From peptide 1b, 2b to 3b, the helicity declined gradually,
FITC-βA-R[cyclo-CILHS
FITC-βA-R[cyclo-CILHS
FITC-βA-R[cyclo-CILHS
(2-Ph)]LLQDS-NH
(2-Bn)]LLQDS-NH
(2-H)]LLQDS-NH
2
2
which may suggest branched substitution groups are not
preferred. Similar result was observed with peptide 5b, which
was even less helical. Notably, peptide 4b was observed to be
5
5
2
FITC-βA-R[cyclo-CILHS
5
Fig. 4 (A) Schematic presentation of fluorescence polarization (FP) assay. (B)
more helical and the rigid planar shape of phenyl may account Binding of 10 nM peptide FITC-1a/b with ER-α at 20°C. Buffer system, 10μM 17-β-
estradiol, 20mM Tris-HCl pH 8.0, 25mM NaCl, 10% glycerol and 1mM TCEP. mP,
for the enhancement.
mean
± s. d. and n = 3. Non-linear regression analysis by Origin 8.0 (C)
2
| J. Name., 2012, 00, 1-3
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Relationship between peptides’ helicity and binding affinity. (D) Binding affinity of
peptide FITC-1b-5b and FITC-6.
As a conclusion, the substitution groups on the in-tether
chiral centre of CIH peptides showed significant influences on
View Article Online
DOI: 10.1039/C6OB02289H
The peptides’ cellular uptakes and stability were then
tested. Flow cytometry analysis was utilized to quantify the
cellular uptakes of peptides in Hela cells. The results clearly
backbone peptides’ helical contents, target binding affinity and
cellular uptakes. This proof-of-concept study unambiguously
showed that the CIH strategy provides a valuable in-tether
modification site without alterations in the backbone peptides.
This modification site could be utilized for various
modifications, biological applications and SAR studies of
peptide therapeutics. In addition, the CIH strategy provides a
unique way to study the function differences casted by soly
conformational differences between peptide epimers. Further
investigation of the in-tether modification site is undergoing in
the laboratory and will be reported in the due course.
indicated that peptide
uptakes than their
increment as shown in Fig. 5A. Confocal microscopy imaging
showed peptide epimers distributed diffusely into cell and
b
epimers showed better cellular
a
counterparts, with a 1.2-2.5 folds
b
the majority of peptides was localized in the cytoplasm.
Notably, a significant fraction of peptide 4b was also detected
in the nucleus (Fig. 5D and Fig. S3). For peptide 1-5b, their
permeability correlated relatively well with their helical
contents as shown in Fig. 5B. Again, peptide
shown in Fig. 5A and B. The relatively poor cellular uptake of
peptide may be explained by the tether hydrophobicity This work is supported by the Natural Science Foundation of China
difference. However, a conclusive elucidation of why peptide Grants 21372023 and 81572198; MOST 2015DFA31590, the
6 is an outliner as
6
6
’s high helical contents didn’t improve its target binding Shenzhen Science and Technology Innovation Committee
JSGG20140519105550503, JCYJ20150331100849958,
JCYJ20150403101146313, JCYJ20160301111338144,
JCYJ20160331115853521 and JSGG20160301095829250; the
Shenzhen Peacock Program KQTD201103.
affinity and cellular uptake is still absent and more persuasive
assays may need be developed for explanation.
The cyclic peptides’ stability was significantly enhanced
comparing with their linear analogue. Per the in vitro serum
stability assay shown in Fig. 5C, linear peptide precursor of
peptide 6, 6-linear degraded in a few hours, while peptides 1b
-
5
b
and
6
remained more than 60 percent intact after 24 hours.
Notes and references
D
FITC
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Fig. 5 Permeability and stability of peptides. (A) Flow cytometry measurements of
Hela cells with 5 μM FITC-labelled peptides at 37 ℃ for 2h. (B) Relationship
between peptides’ helicity and permeability. (C) Serum stability measurements
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s. d. and n = 3. (D) Fluorescent confocal microscopy images of Hela cells treated
with 5 μM FITC-labelled peptides at 37 ℃ for 4h (DNA, blue (DAPI) peptides,
green (FITC)).
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C6OB02289H
Different In-tether chiral centre show distinguishable properties proving In-tether
chiral centres are valuable modification site for constructing peptide ligands.