Design of triazole-stapled BCL9 α-helical peptides to target the β-catenin/B-cell CLL/lymphoma 9 (BCL9) protein-protein interaction

Article abstract of DOI:10.1021/jm201125d

The interaction between β-catenin and B-cell CLL/lymphoma 9 (BCL9), critical for the transcriptional activity of β-catenin, is mediated by a helical segment from BCL9 and a large binding groove in β-catenin. Design of potent, metabolically stable BCL9 peptides represents an attractive approach to inhibit the activity of β-catenin. In this study, we report the use of the Huisgen 1,3-dipolar cycloaddition reaction to generate triazole-stapled BCL9 α-helical peptides. The high efficiency and mild conditions of this "click" reaction combined with the ease of synthesis of the necessary unnatural amino acids allows for facile synthesis of triazole-stapled peptides. We have performed extensive optimization of this approach and identified the optimal combinations of azido and alkynyl linkers necessary for stapling BCL9 helices. The unsymmetrical nature of the triazole staple also allowed the synthesis of double-stapled BCL9 peptides, which show a marked increase in helical character and an improvement in binding affinity and metabolic stability relative to wild-type and linear BCL9 peptides. This study lays the foundation for further optimization of these triazole-stapled BCL9 peptides as potent, metabolically stable, and cell-permeable inhibitors to target the β-catenin and BCL9 interaction.

Full text of DOI:10.1021/jm201125d

Article
pubs.acs.org/jmc
Design of Triazole-Stapled BCL9 α-Helical Peptides to Target the
β-Catenin/B-Cell CLL/lymphoma 9 (BCL9) Protein−Protein Interaction
Steven A. Kawamoto,†,‡ Adriana Coleska,‡ Xu Ran,† Han Yi,† Chao-Yie Yang,† and Shaomeng Wang*,†,‡
†
‡
Departments of Medicinal Chemistry, Internal Medicine and Pharmacology, Comprehensive Cancer Center,
University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: The interaction between β-catenin and B-cell
CLL/lymphoma 9 (BCL9), critical for the transcriptional
activity of β-catenin, is mediated by a helical segment from
BCL9 and a large binding groove in β-catenin. Design of
potent, metabolically stable BCL9 peptides represents an
attractive approach to inhibit the activity of β-catenin. In this
study, we report the use of the Huisgen 1,3-dipolar
cycloaddition reaction to generate triazole-stapled BCL9 α-
helical peptides. The high efficiency and mild conditions of this “click” reaction combined with the ease of synthesis of the
necessary unnatural amino acids allows for facile synthesis of triazole-stapled peptides. We have performed extensive optimization
of this approach and identified the optimal combinations of azido and alkynyl linkers necessary for stapling BCL9 helices. The
unsymmetrical nature of the triazole staple also allowed the synthesis of double-stapled BCL9 peptides, which show a marked
increase in helical character and an improvement in binding affinity and metabolic stability relative to wild-type and linear BCL9
peptides. This study lays the foundation for further optimization of these triazole-stapled BCL9 peptides as potent, metabolically
stable, and cell-permeable inhibitors to target the β-catenin and BCL9 interaction.
The interaction between BCL9 and β-catenin proteins is
mediated by an approximately 25-residue helical segment from
BCL9 and a large binding groove in β-catenin, and design of
nonpeptide, potent small-molecule inhibitors to block this protein−
protein interaction (PPI) would be expected to be difficult. Indeed,
no such small-molecule inhibitors have been reported to date. An
alternative strategy to target this PPI is to design and synthesize
stapled helical peptides based upon BCL9. In the past few years, it
has been demonstrated that stapled helical peptides, designed to
induce conformational constraint in the side chains of the helical
peptide residues, may lead to compounds with enhanced cell-
permeability and metabolic stability.4
One of the most notable methods of helix stabilization has been
the use of ring-closing olefin metathesis (RCM) to link the side
chains of the i and i+4 or i+7 positions (Figure 1A).5 This method
of producing hydrocarbon “stapled” α-helical peptides, introduced
first by Blackwell and Grubbs,5a and further refined by Verdine et
al.,5b has been shown to increase helical propensity and peptide
binding proclivity while decreasing the rate of peptide cleavage by
proteases. Furthermore, these hydrocarbon stapled peptides have
been shown to have cellular activity in several biological settings.6
More recently Cantel et al. demonstrated that the Cu(I)-mediated
Huisgen 1,3-dipolar cycloaddition reaction7 (aka a “click” reaction)
can be used to generate a 1,4-substituted 1,2,3-triazole between side
chain azido and alkynyl moieties at the i and i+4 positions of a pep-
tide chain (Figure 1B) that effectively mimics the use of the
INTRODUCTION
■
Upregulation of β-catenin, the primary mediator of the Wnt
signaling pathway, plays an important role in the tumorigenesis
of several types of human cancer, including colon cancer, pro-
state cancer, and melanoma.1 Functioning as a transcriptional
activator, β-catenin controls the expression of a number of key
genes that regulate the cell cycle and apoptosis. Transcriptional
activation mediated by β-catenin requires the formation of a
β-catenin supercomplex, consisting of B-cell lymphoma 9 (BCL9),
BCL9-like (B9L), a T-cell factor/lymphoid enhancer factor (TCF/
LEF), the cAMP response element-binding protein (CBP), and
other cofactors.2 It has been proposed that small-molecule inhibitors
capable of disrupting the protein−protein interactions of β-catenin
and its key cofactors may be a promising strategy to block β-catenin
activity.2 Previous efforts have indeed led to the successful
identification of several classes of small-molecule inhibitors that
are capable of disrupting the interactions of β-catenin with TCF or
CBP, thereby inhibiting β-catenin-mediated transcriptional activa-
tion.2 Some of these inhibitors also cause inhibition of cell growth in
cancer cells with constitutively activated β-catenin.2
Analysis of the recently reported crystal structure of β-cate-
nin in a complex with BCL9 and TCF showed that the interac-
tion between β-catenin and BCL9 is distinct from that of other
β-catenin binding partners3 and is mediated by a well-defined
binding groove in β-catenin and several hydrophobic and charged
residues from the α-helix of BCL9. This binding interaction, with a
Kd value of 0.5 μM, suggests that the β-catenin−BCL9 interaction
may be an attractive site for the design of potent and specific
small-molecule inhibitors capable of blocking β-catenin activity.
Received: August 22, 2011
Published: December 23, 2011
© 2011 American Chemical Society
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Figure 1. Comparison of helix stapling techniques. (A) Hydrocarbon stapling. Peptides are synthesized on resin, and the S-olefin-containing amino
acids are incorporated at the i and i+4 positions. The olefins are then connected by means of an on-resin RCM reaction and then the peptide is
cleaved and purified. For clarity, only i/i+4 stapling is depicted. (B) Triazole stapling. Peptides are synthesized on resin, and the azido and alkynyl
amino acids are incorporated at the i and i+4 positions. The peptide is then cleaved and cyclized in solution using the Huisgen Cu(I)-mediated 1,3-
dipolar cycloaddition reaction.
analogous lactam bridge.8 This cycloaddition reaction has been
popularized by Sharpless and co-workers in the past decade and
has been called a “click” reaction due to its simplicity, high yields,
mild reaction conditions, and biocompatibility.9 The use of
cheaper, less toxic copper reagents also makes click-mediated
stapling an attractive alternative to ruthenium-based RCM-type
cyclization methods. Unlike hydrocarbon stapling, however,
this method of triazole stapling has not been rigorously tested
and its influence on peptide helicity and protease resistance has
not been examined to date.
In the present study, we employed the triazole stapling method
to stabilize BCL9 α-helical peptides for targeting the BCL9/
β-catenin protein−protein interaction. Furthermore, in the process
(PDB code: 2GL7) of BCL9 (gray) in complex with β-catenin (cyan)
Figure 2. Solvent-exposed residues in BCL9. The crystal structure
of developing triazole-stapled BCL9 peptide inhibitors, we
optimized the linker lengths, staple orientation, and amino acid
stereochemistry required for generation of the optimal triazole
staples in our peptides. We also investigated various stapling sites
and successfully generated double triazole-stapled peptides that
exhibit over 90% helicity and improved protease resistance.
shows that L356, E360, Q364, and D368 (colored magenta) are
exposed to solvent with Q364 having the only fully resolved side chain.
To maintain a high net positive charge for the stapled peptide, solvent-
exposed arginine residues were not considered for stapling sites. The
BCL9 sequence is shown at the top with the corresponding solvent-
exposed residues colored magenta. E360 and Q364 are numbered.
for use in triazole stapling (Figure 2). These residues were both
exposed to solvent and located in the central region of our
optimized BCL9 24-mer peptide.11 Thus, a triazole staple between
these positions would be predicted to have a significant stabilizing
effect on both the N- and C-terminal portions of the peptide.
Other solvent exposed residues were also examined, but arginine
residues were omitted from consideration so as to maintain an
overall positive charge for our triazole-stapled peptides. Verdine et
al. have demonstrated that the likelihood that stapled peptides will
be cell permeable are increased when the stapled peptide has a net
neutral or positive charge.6a
Before introducing a triazole staple, we used computational
modeling to examine the different combinations of linker
lengths and isomers for use in the staple. Various combinations
of L-Nle(εN3) or L-norvaline(δN3) [Nva(δN3)] and L- or D-Pra
linkers were examined, and the locally minimized structures of
the triazole-stapled peptides were compared with the BCL9
RESULTS
■
In contrast to hydrocarbon stapling, which has been extensively
optimized and utilized in a number of different model sys-
tems,5b,6a−c,10 the triazole stapling method has not been well
studied and there has been only limited investigation into the
optimal length of the linker, the order of the azide and alkynyl
residues, or the use of L versus D enantiomers. Cantel et al.
demonstrated that cyclization of L-norleucine(εN3) [i.e.
Nle(εN3)] and L-propargylglycine (Pra) substituted at the i
and i+4 positions, respectively, mimicked the amide-stapled
peptide inhibitor of parathyroid hormone-related peptide
(PTHrP), albeit with minor differences in the peptide backbone.8
Using this combination as a starting point, we systematically ex-
amined the effect of linker length, position of the triazole ring within
the staple, and use of different stereoisomers for the staple.
Analysis of the crystal structure of BCL9 in a complex with
β-catenin indicated that E360 and Q364 were suitable residues
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Figure 3. Molecular modeling of possible triazole-stapled BCL9 peptides. Top: The BCL9 24-mer. Highlighted in red are the residues replaced by
azido and alkynyl amino acids (magenta *). (A−D) Locally minimized structures of triazole-stapled BCL9 peptides using various combinations of L-
Nle(εN3) or L-Nva(δN3) and L- or D-Pra. The combinations of unnatural amino acids used are given above each structure. The BCL9 peptide from
the reported cocrystal structure with β-catenin (PDB code: 2GL7) is shown in yellow. Locally minimized BCL9 peptides are shown in green with the
triazole staple highlighted in magenta/blue.
peptide from the cocrystal structure (Figure 3). As shown in
Figure 3A−C, using only L-amino acids or shortening the length
of the staple by using Nva(δN3) caused noticeable changes in the
backbone conformation. On the other hand, the L-Nle(εN3)/D-Pra
combination of residues at the E360 and Q364 (i and i+4, respec-
tively) positions showed the least amount of distortion in the
peptide backbone (Figure 3D). Thus, our first triazole-stapled
peptides incorporated L-Nle(εN3) in place of E360 and either L- or
D-Pra in place of Q364.
To synthesize the requisite Fmoc-protected L-Nle(εN3)-OH
residue, we converted Fmoc-L-Lys-OH to its azide derivative in
one step according to the reported method.12 Cyclization of the
peptides was monitored by analytical RP-HPLC, with the cycli-
zed product typically having a retention time differing from the
linear precursor peptide by approximately 0.1−0.6 min (Figure S1,
Supporting Information). We found that the conditions of Cantel
et al.,8 which employed 4.4 equiv of CuSO4 and ascorbic acid,
and a peptide concentration of 1 mg/mL in 2:1 H2O/t-BuOH,
worked well for our system. As evidence of the ease of this click
reaction, we observed that reaction overnight as previously reported
was unnecessary as the reaction appeared to be complete within
20−60 min at room temperature. We did observe partial oxidation
of our BCL9 peptides, probably at M372. Mutation of M372 to Leu
(the corresponding residue in the B9L homologue; peptide 2 in
Table 1) in subsequent experiments eliminated this problem and
did not affect the binding affinity of our peptides.
Using these reaction conditions, we synthesized two sets of
triazole-stapled peptides bearing either L- or D-Pra at position
364 (11 and 12, respectively, in Table 1). The L-Nle(εN3) and
L-Pra combination gave a linear precursor peptide 3 with a
binding affinity to β-catenin half that of the wild type BCL9 24-mer
(Table 1). In contrast, the triazole-stapled peptide 11 was twice as
potent as the wild type peptide 1 and 4 times more potent than
its linear precursor 3. The linear precursor peptide 4 with the
L-Nle(εN3) and D-Pra combination, was 8-fold weaker than the
wild type, which was expected as a result of the introduction of a
D-amino acid into the sequence. However, the triazole-stapled
peptide 12 was 4-fold more potent than the wild type BCL9
24-mer and 38-fold more potent than its linear precursor. These
results supported our computational modeling which suggested that
the use of D-Pra may cause less distortion of the α-helical peptide
backbone in the triazole-stapled peptide.
Circular dichroism (CD) spectroscopy confirmed that the
triazole-stapled peptides were indeed more helical than the
linear wild-type peptide 1 or their linear precursors 3 and 4.
Peptide 11, which utilizes all L-amino acids, showed an improve-
ment to 66% helical content in PBS compared to 44−45% for the
wild-type peptide 1 and the uncyclized peptides 3 and 4 (Figure 4).
Peptide 12, which incorporates the D-Pra at the i+4 position of the
staple, was determined to be 90% helical. Although the helicity
for 12 seemed unusually high, it was correlated with its higher
binding affinity than 11.
Baldwin and co-workers showed that far-UV CD spectrum of
a peptide can be affected by an aromatic group within the
peptide, thus yielding a false enhancement of helical propensity
of the peptide.13 To study the effect of 1,2,3-triazole aromatic
ring to CD spectrum and helical propensity, we synthesized
unstapled peptides 31 and 32, which contain a triazole ring
based upon peptides 11 and 12, and analyzed their CD spectra
(Figure S2, Supporting Information). Our data showed that the
CD spectra for peptides 31 and 32 are very similar to those of
peptides 3 and 4 without the triazole ring. The calculated
helical propensities for peptides 3, 4, 31, and 32 are also very
close (Figure S2, Supporting Information). These data thus
further confirmed that the triazole-staple strategy via cyclization
is successful in enhancing the helical conformations for BCL9
peptides.
We next sought to determine the optimal length of the
triazole staple and the position of the triazole within the staple,
and to address these questions, synthesized the azido derivative
of Fmoc-L-ornithine [Fmoc-L-Nva(δN3)], which is one carbon
shorter than the lysine used in the synthesis of Fmoc-Nle(εN3).
In addition, we used a Ni(II) complex of a glycine Schiff’s base
with (S)-2-[N,(N′-benzylprolyl)amino]benzophenone (BPB;
Scheme 1) to synthesize propargylalanine (Paa), which is one
carbon longer than Pra.14 Issad et al. recently reported on the
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d
Table 1. Optimization of Linkers for Triazole Stapling of BCL9 Peptides
a
b
Ki values were determined using a BCL9-competitive FP binding assay. Ki value was extrapolated from an incomplete binding curve due to poor
c
d
solubility. Peptide too insoluble for accurate measurement. *, L-amino acid; #, indicates D-amino acid; NB, no binding; ND, not determined.
use this complex for accessing a range of amino acids that can
be used for triazole stapling.8 The starting complex is relatively
simple to synthesize on large scale, and its use for the synthesis
of monosubstituted amino acids is straightforward and gives
acceptable yields.
With several different azido and alkynyl amino acids in hand,
we systematically examined the effect of shortening or length-
ening the staple, moving the triazole within the staple, or
reversing the direction of the triazole staple (Table 1). All linear
uncyclized peptides had low micromolar binding affinities
(peptides 5−10), but when the peptides were cyclized, significant
differences became apparent. Shortening the linker by one carbon
(13) resulted in complete loss of binding affinity for β-catenin and a
reduction in helical structure. Lengthening the linker by one carbon
(14) also resulted in weaker binding affinity (2-fold weaker than
uncyclized, 5-fold weaker than BCL9 24-mer 1) and showed no
helix stabilizing effect. In addition, shifting the position of the tri-
azole by one atom (15) markedly reduced the binding affinity (50-
fold weaker than BCL9 24-mer) and also resulted in significantly
worse peptide solubility. Shifting of the triazole to a more central
position in the staple also perturbed the helical structure of the
peptide, indicating that the dipole of the triazole itself may have an
influence on the peptide structure. Reversing the linker did not
significantly affect the binding affinity when using both L-Pra and
L-Nle(εN3) (16), but the solubility of the resulting peptide was
decreased. The reversed staple could also tolerate use of the D-form
of the Nle(N3) at the i+4 position (17), but using D-Pra at position
360 (18) resulted in 6-fold weaker binding affinity.
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the peptide. For 24, we speculated that the drop in helicity is
associated with the reversed orientation and triazole staple and
possibly a different effect of the triazole dipole with the peptide itself.
This same modest, but noteworthy drop in binding affinity and
helical content was also seen in the reversed staples at the original
position (compare peptides 16 and 17 to 11 and 12). Nevertheless,
we confirmed three potential stapling sites in our BCL9 peptide and
demonstrated that either orientation of the triazole staple could be
used, albeit with a preference for the azide/alkyne orientation at the
i/i+4 positions over the reverse orientation.
On the basis of the data obtained from the peptides stapled
at different positions, we proposed that a double stapled peptide
may further improve binding affinity and helical content and
possibly metabolic stability. In contrast to hydrocarbon stapling,
we hypothesized that the unsymmetrical nature of the triazole
staple would allow for two staples to be placed adjacent to one
another on consecutive turns of the helix, simply by reversing the
orientation of the second staple. Having the two staples in oppo-
site orientations would effectively put incompatible linkers (i.e.,
both alkynes or both azides) from each staple adjacent to each
other and reduce the chance of “mixed staples” (Figure 5). It
should be noted, however, that Verdine et al. recently demon-
strated that hydrocarbon stapling between the i and i+3 or i+4
positions could be used to generate double hydrocarbon staples in
a polyalanine model peptide when the staples were separated by a
two residue spacing.10a This short spacing was shown to place the
two adjacent olefins (one from each staple) in an unfavorable
position to generate mixed staples. However, in our peptide, the
majority of staple locations that fail to interfere with binding inter-
actions or reduce the number of positively charged residues occur
exactly at the i and i+4 positions relative to each other. These posi-
tions may thus lead to significant amounts of mixed stapling when
using the hydrocarbon stapling procedure.
To generate the first double triazole-stapled BCL9 peptide,
we combined the staple combinations and positions used in 20
and 24 to synthesize 26 (Table 2). Reversing both staples
allowed us to synthesize 28. Both 26 and 28 showed reasonable
binding affinity, and their helical percentages were increased
to >90% (Table 2). Because we suspected that the reverse
triazole staples (i.e., L-Pra/D-Nle(εN3)) might be causing a slight
decrease in the potency of our peptides, we synthesized 30 in
which the triazole staples were spaced out by one full turn of
the helix. This additional spacing allowed for both staples to
utilize the optimal L-Nle(εN3)/D-Pra linker combination and
resulted in a 2−3-fold improvement in the binding affinity of
30 which also had a helical content >95%.
Figure 4. CD spectra of triazole-stapled BCL9 peptides. Peptides were
dissolved in PBS, pH 7.4, at a final concentration of approximately
50 μM. Exact peptide concentrations were determined using the BCA
Protein Assay Kit. The spectra were averaged over 10 scans. Percent
helicity was calculated from the mean residue ellipticity, θ, at 222 nm.
We thus concluded that for triazole stapling of one turn of an
α-helix, the optimal staple consists of an 8-atom linker consist-
ing of L-Nle(εN3) and D-Pra at the i and i+4 positions, respec-
tively. Although the use of the D-Pra at the i+4 position was
only marginally better than use of L-Pra, the incorporation of
the D-amino acid served as a negative control for peptide cy-
clization. Unlike the analogous RCM reaction in which the
linear and stapled peptides differ in mass by 28 (C2H4), the
triazole-stapled peptides have molecular weights that are
identical to their linear uncyclized precursors. Without NMR
or IR spectra to confirm the formation of the triazole ring, the
successful triazole stapling was deduced from both the shift in
retention time in the analytical RP-HPLC chromatogram (Figure S1,
Supporting Information) and the 10−38-fold difference in binding
affinities between the linear and triazole-stapled peptides (compare 4
and 12, 9 and 17).
We then investigated different stapling sites within our BCL9
24mer peptides (Table 2). Because this 24mer peptide is fully
helical with six complete helical turns, there were a number of
positions that could be used for stapling, several of which are
illustrated in Figure 2. Using the optimal L-Nle(εN3)/D-Pra
combination of linkers, we positioned the triazole staple closer
to the N-terminus (19 and 20), or closer to the C-terminus (21
and 22), but found little difference in binding affinity. The peptide
could also tolerate the reverse staple toward the C-terminus, again
with little change in binding affinity (24). Although the C-terminal
stapled peptide 22 retained a high degree of helical character, there
was a decrease in the helical content of 20 and 24. In the case of
20, we hypothesized that the Thr residue, which is known to
disfavor helical structure,15 might be the reason for this decrease in
helicity and that positioning a single staple away from this Thr
residue was insufficient to maintain helical structure throughout
Finally, we tested whether these double triazole-stapled peptides
with a very high helicity would be resistant to proteolytic degradation.
Using cell media taken from actively cultured SW480 colon cancer
cells, we examined the rate of proteolysis of the wild-type peptide 2
and of two double triazole-stapled BCL9 peptides 28 and 30. The
Scheme 1. Synthesis of Fmoc-L-propargylalanine (Paa)
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b
Table 2. Optimization of Triazole Stapling Location and Number of Staples for BCL9 Peptides
double triazole-stapled peptides 28 and 30 both demonstrated a
markedly improved stability over the linear peptide 2 (Figure 6).
disulfide linkages, amide linkages, and hydrocarbon linkages.4
Verdine’s group demonstrated that RCM between side chain
olefins at the i and i+4 or i+7 positions could be used to generate
hydrocarbon staples that significantly improve the helical character
of peptides in aqueous solution.5b This method of hydrocarbon
stapling has been shown to increase peptide binding affinity, cell
permeability, and protease resistance,5b,6a,c,d,16 but it requires the
multistep synthesis of the requisite olefin amino acids and uses the
fairly toxic ruthenium catalysts.
Triazole stapling was recently introduced by Cantel et al. and
shown to be a good replacement for a lactam bridge between
the i and i+4 positions of a peptide helix.8 In addition, the
Huisgen 1,3-dipolar cycloaddition reaction employed to
generate the triazole staple uses inexpensive and nontoxic
reagents and the requisite azido and alkynyl amino acids are
DISCUSSION
■
Design of nonpeptide, small molecules to inhibit protein−protein
interactions (PPIs) is a major challenge in modern drug discovery
due to large surface area of the interactions between proteins. For
this reason, peptide-based compounds can serve as an alternative for
targeting PPIs. In the past, the use of peptides has been limited to
cell-free in vitro experiments due to their poor stability and cell
permeability. To overcome these major issues, researchers have
focused on the design of conformationally restricted peptides that
stabilize the secondary structure of peptides and improve their
metabolic stability. To stabilize α-helical peptides, a number of
approaches have been investigated including the use of helix caps,
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peptides regained their potency only after the successful completion
of the triazole staple.
As with any type of “stapling” method, the optimal location of
the staple within the peptide sequence needs to be determined
experimentally. Examination of the crystal structure of BCL9 in a
complex with β-catenin (PDB code: 2GL7) allowed identification
of several combinations of solvent exposed residues that could be
replaced in order to generate the triazole staple. As additional con-
siderations for selection of a stapling site, we attempted to maximize
the substitution of acidic residues and avoided the replacement of
basic residues so as to increase the overall positive charge of the
peptide. Bernal et al. demonstrated that the probability of cell
permeability of hydrocarbon-stapled peptides was improved when
the peptide had an overall neutral or positive charge.6a Accordingly,
we examined three different locations and found that either the
central position (residues 360 and 364) or the C-terminal position
(residues 364 and 368) were effective in stabilizing the helical
structure of the peptide and in maintaining potent binding affinity.
We hypothesize that these two locations work well due to their
close proximity to T365. Threonine residues are known to disfavor
helical structure,15 and this residue may possibly cause the
“unraveling” of the peptide helix when the staple is located some
distance away.
Figure 5. Double triazole-stapled peptides. Reversing the order of the
azido and alkynyl linkers favors the generation of a double triazole
staple (left) and discourages the formation of mixed staples (right).
We next explored the design of double triazole-stapled peptides.
The synthesis of a double hydrocarbon-stapled polyalanine peptide
was recently demonstrated, but it requires that the two staples
be separated by only two residues.10a In our case, the only suitable
stapling sites that did not require mutation of an arginine residue
were spaced all at exactly the i and i+4 positions relative to each
other, making hydrocarbon stapling problematic. In contrast,
triazole stapling uses unsymmetrical linkers so by reversing the
orientation of one staple relative to the other, we were able to
generate double triazole-stapled peptides with a minimal amount of
mixed stapling. These double triazole-stapled peptides showed
binding affinities comparable to most of the single stapled peptides;
however, their helical content was increased to over 90% in aqueous
solution. We also found that separating the staples by one and a half
turns of the helix also allowed us to generate a double triazole-
stapled peptide with increased potency and over 90% helicity. We
hypothesized that in longer peptides, those having more than four
helical turns, a single triazole staple may be inadequate to prevent
the unraveling of the ends of the peptide. However, by utilizing two
strategically placed staples these longer peptides can be stabilized
much more effectively, which will increase their proteolytic stability
and potentially also their cell permeability. Indeed, we found that in
addition to one of the single stapled peptides, two of our double-
triazole stapled peptides (28 and 30) showed a marked improve-
ment in its stability in cell media. Studies are currently in progress to
evaluate the activity of these triazole-stapled BCL9 peptides in cell-
based assays. If these peptides prove to be cell permeable, they will
serve as useful biological tools with which to evaluate the true effects
of inhibiting the interaction of BCL9 with β-catenin.
Figure 6. Stability of linear and double triazole-stapled BCL9 peptides.
Peptides were diluted in cell culture media (Leibovitz-15, 10% FBS,
50 U/mL penicillin and streptomycin) taken from actively cultured
SW480 cells and incubated at 37 °C. Aliquots were taken at various
time points, quenched with 1% TFA/MeOH, and analyzed by LC-MS.
easily obtained from commercial sources or can be synthesized
in one step. However, this method of triazole stapling has not
been extensively studied and its effects on peptide binding
affinities, helical propensity, metabolic stability, and cell
permeability have not been addressed.
In the present study, we employed the use of triazole stapling
to generate stabilized α-helical BCL9 peptides as inhibitors of
the BCL9/β-catenin binding interaction. In the process, we
performed a detailed study of the optimal combinations of
azido and alkynyl linkers. After examining a range of linkers, we
found that for stabilization of one turn of the helix the optimal
triazole-containing staple was eight atoms in length. Further-
more, the helical propensity of the peptide was extremely dependent
on the position of the triazole within the staple, with the preferred
staple having the triazole ring off-center. In addition, there appeared
to be a slight preference for the azido and alkynyl amino acids to be
at the i and i+4 positions, respectively, instead of in the reverse ar-
rangement. Our computational modeling, binding studies, and CD
studies also indicated a preference for the use of D-Pra rather than
the L-Pra at the i+4 position. In addition, use of D-Pra also provided
an important negative control for the cyclization reaction because
completion of the reaction could not be determined by mass spec-
trometry due to the same molecular weight before and after the
reaction. Introducing D-Pra into the peptide caused a significant
decrease in the activity of the linear precursor peptide, and the
CONCLUSION
■
In this study, we performed extensive optimization of the triazole
stapling strategy to generate single and double triazole-stapled
BCL9 peptides. Our data showed that the combination of
L-Nle(εN3) and D-Pra substituted at the i and i+4 positions,
respectively, produced the best results for generation of single
triazole-stapled peptides. These peptides demonstrated increased
potency and improved helical structure. In addition, double-
stapled peptides were over 90% in helical propencities and demon-
strated potent binding to β-catenin. Several of the designed single
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synthesizer unless otherwise indicated. L- and D- enantiomers of Fmoc-
Pra-OH were purchased from Advanced Chemtech or Chem-Impex
International, Inc. All peptides were cleaved and deprotected with
87.5% TFA, 5% dithiothreitol, 5% H2O, 2.5% triisopropylsilane, and
then purified by semipreparative RP-HPLC using a Waters Sunfire
19 × 150 C18 column followed by lyophilization for 16−24 h. Mass
spectrometry was performed with a Thermo-Finnigan LCQ Deca or
LCQ Fleet electrospray ionization mass spectrometer. Purity of all the
synthesized peptides was determined by HPLC to be >95%, and such
information is provided in Table S1 in the Supporting Information.
Triazole-Stapled Peptides. General Procedure. Pure, lyophi-
lized linear peptide and CuSO4·5H2O (4.4 equiv) were dissolved in 2:1
H2O/t-BuOH to give a final peptide concentration of 1 mg/mL. Alter-
natively, hydrophobic peptides can be dissolved in a minimum amount
of 6 M guanidine hydrochloride solution and then added dropwise to
the CuSO4/H2O/t-BuOH solution to prevent peptide precipitation.
Sodium L-ascorbate (4.4 equiv) dissolved in H2O (2 mL) was added
slowly. The reaction was stirred at room temperature for 30−90 min.
The reaction was then concentrated in vacuo and purified by semi-
preparative RP-HPLC. Typical yields of cyclized product were 40−
60%. Triazole-stapled peptides typically had retention times
approximately 0−0.6 min longer than their linear, unstapled precursor.
Modeling of Triazole Staples. A helical peptide segment of
BCL9 (residues S352 to F374) taken from the crystal structure of
BCL9 in a complex with β-catenin (PDB code 2GL7) was used as a
template with which to construct stapled peptides. Triazole staples of
varying lengths, orientations, and stereochemistries (at the peptide
backbone) were constructed. After the staple was formed, structures
were locally minimized using Sybyl (Tripos, Inc., St. Louis, MO). The
locally minimized structures were then superimposed on the original
BCL9 peptide segment obtained from the crystal structure.
Fluorescence Polarization (FP) Competitive Binding Assays.
Peptide binding affinities for β-catenin were determined using our
optimized BCL9-competitive FP binding assay and the BCL9-derived
fluorescent probe 8-F described previously.11
(CD Experiments. CD measurements were performed at room
temperature (20 °C) using a Jasco J-715 and a quartz flow cell with a
1 mm path length. Peptides were dissolved in phosphate buffered saline
(PBS, pH 7.4) at a concentration of approximately 0.17 mg/mL.
Precise peptide concentrations were then determined using the BCA
Protein Assay Kit (Pierce) according to the manufacturer’s protocol.
Spectra were collected at 50 nm/min, using a bandwidth of 1 nm,
averaged over 10 scans, and the baseline (PBS only) was subtracted
from each spectrum. The mean residue ellipticity [θ]222 at 222 nm was
calculated using eq 1,
and double triazole-stapled peptides also showed improved resistance
to proteolytic degradation. Finally, we showed that triazole stapling
can be used to generate double-stapled peptides in scenarios in
which hydrocarbon stapling could lead to significant amounts of
mixed stapling. Hence, triazole stapling can be an attractive approach
for the design of stabled α-helical peptides with improved binding
affinities toward their molecular targets, as well as improved metabolic
stabilities.
EXPERIMENTAL SECTION
■
General Synthesis of Fmoc-azido Amino Acids. Fmoc-
Nle(εN3)-OH and Fmoc-L-Nva(δN3)-OH were synthesized from
Fmoc-Lys-OH and Fmoc-L-Orn(Boc)-OH, respectively, according to
the previously reported protocol.12 The L- and D- enantiomers of
Fmoc-Nle(εN3)-OH along with Fmoc-L-Orn(Boc)-OH were pur-
chased from Advanced Chemtech or ChemImpex International, Inc.
Propargylalanine (Paa)-Ni-BPB. (S)-Gly-Ni-BPB14b (2.78 mmol)
and crushed NaOH (27.8 mmol) were suspended in anhydrous DMF
under vacuum. The flask was purged with nitrogen, and the reaction was
stirred at room temperature for 7 min. 4-Bromo-1-butyne (3.83 mmol)
was added via syringe, and the reaction was stirred for an additional 6 min,
then poured into 5% acetic acid (120 mL) and extracted with toluene
(3 × 40 mL). The combined organic extracts were washed with brine
(20 mL), dried over MgSO4, filtered, concentrated in vacuo, and purified
by flash column chromatography (4:1 ethyl acetate/acetone) to afford the
product as a red solid (1.94 mmol; 70%). Rf 0.3 (4:1 EtOAc/acetone);
20
1
[a]D = +2390° (c = 0.024, CHCl3). H NMR (300 MHz, CDCl3)
δ 8.13 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 6.9 Hz, 2H), 7.56−7.46 (m, 3H),
7.36 (t, J = 7.5 Hz, 2H), 7.28−7.12 (m, 3H), 7.01−6.99 (m, 1H), 6.70−
6.61 (m, 2H), 4.45 (d, J = 12.6 Hz, 1H), 3.99 (dd, J = 8.7, 3.6 Hz, 1H),
3.70−3.60 (m, 1H), 3.59 (d, J = 12.6 Hz, 1H), 3.54−3.44 (m, 2H), 2.81−
2.75 (m, 2H), 2.60−2.49 (m, 1H), 2.46−2.02 (m, 4H), 1.90−1.83
(m, 1H), 1.80 (t, J = 2.7 Hz, 1H). 13C NMR (75.5 MHz, CDCl3)
δ 180.34, 178.80, 171.04, 142.26, 133.71, 133.33, 133.2, 132.26, 131.55,
130.07, 129.74, 129.11, 128.96, 128.94, 128.89, 128.50, 127.85, 127.61,
127.18, 126.35, 123.77, 120.76, 82.65, 70.18, 69.58, 69.38, 63.13, 57.07,
54.04, 34.09, 30.67, 23.90, 15.03. ESI-MS [M + Na]+ calcd for C31H29-
N3NaNiO3 572.15, found 572.27.
Fmoc-Paa-OH. (S)-Paa-Ni-BPB (4.8 mmol) was dissolved in 2:1
MeOH/CH2Cl2 (15 mL) and added dropwise over 10 min to a
refluxing solution of 3N HCl/MeOH at 60 °C. The reaction was
stirred for an additional 20 min and then evaporated to dryness, redis-
solved in H2O (10 mL), and evaporated to dryness again. The crude
residue was dissolved in H2O (30 mL) and washed with CHCl3 (6 ×
20 mL). The combined organic washes were extracted with H2O
(15 mL), and the combined aqueous extracts were neutralized with
Na2CO3. Additional Na2CO3 (15 mmol) was added, and the solution
was cooled to 0 °C. Fmoc-N-hydroxysuccinimide ester (7.5 mmol) in
dioxane (45 mL) was added dropwise, and the reaction was then
allowed to warm slowly to room temperature and were stirred over-
night. The reaction was diluted with H2O (100 mL), acidified with
concentrated HCl, and extracted with EtOAc (3 × 40 mL). The
combined organic extracts were dried (MgSO4), filtered, concentrated
in vacuo, and purified by flash column chromatography (1:1 EtOAc/
hexane, 1% AcOH) to afford the product as a colorless solid. The
product was redissolved in an ether/hexane mixture, concentrated in
vacuo, and dried under high vacuum to remove all traces of AcOH,
leaving the product as a solid (4.5 mmol; 94%). [a]D20 = −14.64° (c =
[θ]
222
= θ /(C·n)
222
(1)
where θ222 is the measured ellipticity at 222 nm, C is the molar
concentration of the peptide, and n is the number of amino acid
residues in the peptide. The maximum mean ellipticity [θ]max was
calculated using eq 2 as reported previously,17
[θ]
max
= ( − 44000 + 250T)(1 − k/n)
(2)
where T is the temperature in degrees Celsius and k is the number of
non-hydrogen-bonded peptide carbonyls.18 For N-terminal acetylated
peptides, k = 3. Percent helicity was then calculated by eq 3,
%helicity = ([θ] /[θ] ) × 100
(3)
222
max
Peptide Stability Assays. Leibovitz-15 cell culture media sup-
plemented with 10% fetal bovine serum and 50 units/mL of penicillin/
streptomycin was used to culture SW480 colon cancer cells. Cells were
cultured for 3 days at 37 °C, in 5% CO2 in a humidified incubator, and then
the cell media was collected and centrifuged at 4000 rpm (900 rcf) for
5 min. The cleared cell culture media was then chilled on ice. Tryptophan
was dissolved in PBS at a concentration of 1 mM and then added to the
cell culture media to give a final tryptophan concentration of 200 μM.
Peptides (20 mM in DMSO) were then added to the cell culture media to
give a final peptide concentration of 200 μM. The peptides were incubated
in cell culture media at 37 °C. Aliquots of the reaction mixture were
1
3.35, MeOH). H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 7.5 Hz,
2H), 7.59 (d, J = 6.9 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (t, J =
7.2 Hz, 2H), 5.40 (d, J = 8.1 Hz, 1H), 4.52−4.39 (m, 3H), 4.23 (t, J =
6.6 Hz, 1H), 2.31−2.28 (m, 2H), 2.20−2.17 (m, 1H), 2.01−1.92 (m, 2H).
13C NMR (75.5 MHz, CDCl3) δ 176.08, 156.11, 143.77, 143.60,
141.34, 127.78, 127.11, 125.05, 120.04, 82.41, 69.87, 67.16, 53.07,
47.14, 30.78, 14.97. ESI-MS [M + Na]+ calcd for C21H19NNaO4
372.12, found 372.13.
General Peptide Synthesis. Automated solid-phase peptide
synthesis was performed on an Applied Biosciences 433A peptide
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Journal of Medicinal Chemistry
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removed at specific time intervals and quenched with three volumes of 1%
TFA in methanol, incubated on ice for 10 min, and then centrifuged at
13000 rpm for a further 10 min. The supernatant was then analyzed by
RP-HPLC-MS using a Surveyor Plus HPLC equipped with a 2.1 mm ×
150 mm Waters C18 Sunfire column and a Surveyor Plus PDA detector
and interfaced with a Thermo Finnigan LCQ Fleet electrospray ionization
mass spectrometer (ESI-MS). Chromatographic separation was achieved
with a flow rate of 0.4 mL/min and a 4.5%/min gradient of solvent B
(acetonitrile, 0.05% TFA, 0.05% AcOH) in solvent A (H2O, 0.05% TFA,
0.05% AcOH). The LC-MS chromatogram was analyzed by integrating the
chromatographic peak for the triply charged parent peptide and dividing it
by the area under the chromatographic peak of the tryptophan internal
control (at 280 nm). The percentage of peptide remaining at each time
point was normalized to the value calculated for the 0 min time point. LC-
MS analysis of each time point was performed in triplicate.
(b) Moellering, R. E.; Cornejo, M.; Davis, T. N.; Del Bianco, C.; Aster,
J. C.; Blacklow, S. C.; Kung, A. L.; Gilliland, D. G.; Verdine, G. L.;
Bradner, J. E. Direct inhibition of the NOTCH transcription factor
complex. Nature 2009, 462, 182−188. (c) Walensky, L. D.; Kung,
A. L.; Escher, I.; Malia, T. J.; Barbuto, S.; Wright, R. D.; Wagner, G.;
Verdine, G. L.; Korsmeyer, S. J. Activation of apoptosis in vivo by a
hydrocarbon-stapled BH3 helix. Science 2004, 305, 1466−1470.
(d) Walensky, L. D.; Pitter, K.; Morash, J.; Oh, K. J.; Barbuto, S.;
Fisher, J.; Smith, E.; Verdine, G. L.; Korsmeyer, S. J. A stapled BID
BH3 helix directly binds and activates BAX. Mol. Cell 2006, 24, 199−
210.
(7) Huisgen, R.; Szeimies, G.; Mobius, L. 1.3-Dipolare Cyclo-
additionen. 32. Kinetik der Additionen organischer Azide an CC-
Mehrfachbindungen. Chem. Ber. 1967, 100, 2494−2507.
(8) Cantel, S.; Isaad Ale, C.; Scrima, M.; Levy, J. J.; DiMarchi, R. D.;
Rovero, P.; Halperin, J. A.; D’Ursi, A. M.; Papini, A. M.; Chorev, M.
Synthesis and conformational analysis of a cyclic peptide obtained via i
to i+4 intramolecular side-chain to side-chain azide-alkyne 1,3-dipolar
cycloaddition. J. Org. Chem. 2008, 73, 5663−5674.
ASSOCIATED CONTENT
* Supporting Information
■
S
Complete listing of peptide sequences, characterization, and
additional proteolytic stability assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
(9) Kolb, H. C.; Sharpless, K. B. The growing impact of click
chemistry on drug discovery. Drug Discovery Today 2003, 8, 1128−
1137.
(10) (a) Kim, Y. W.; Kutchukian, P. S.; Verdine, G. L. Introduction of
all-hydrocarbon i,i+3 staples into alpha-helices via ring-closing olefin
metathesis. Org. Lett. 2010, 12, 3046−2049. (b) Kim, Y. W.; Verdine,
G. L. Stereochemical effects of all-hydrocarbon tethers in i,i+4 stapled
peptides. Bioorg. Med. Chem. Lett. 2009, 19, 2533−2536.
(11) Kawamoto, S. A.; Thompson, A. D.; Coleska, A.; Nikolovska-
Coleska, Z.; Yi, H.; Wang, S. Analysis of the interaction of BCL9 with
beta-catenin and development of fluorescence polarization and surface
plasmon resonance binding assays for this interaction. Biochemistry
2009, 48, 9534−41.
(12) Katayama, H.; Hojo, H.; Ohira, T.; Nakahara, Y. An efficient
peptide ligation using azido-protected peptides via the thioester
method. Tetrahedron Lett. 2008, 49, 5492−5494.
AUTHOR INFORMATION
Corresponding Author
*Phone: (734) 615-0362. E-mail: shaomeng@umich.edu.
■
ACKNOWLEDGMENTS
■
We thank Dr. Ari Gafni of the Department of Biophysics at the
University of Michigan for the use of his CD spectropolarim-
eter. This work is supported in part by the Department of
Homeland Security, University of Michigan Regents, the
American Foundation for Pharmaceutical Education, and the
National Institutes of Health (R21NS066494).
(13) Chakrabartty, A.; Kortemme, T.; Padmanabhan, S.; Baldwin,
R. L. Aromatic side-chain contribution to far-ultraviolet circular dichroism
of helical peptides and its effect on measurement of helix propensities.
Biochemistry 1993, 32, 5560−5565.
ABBREVIATIONS USED
■
BCL9, B-cell CLL/lymphoma 9; B9L, BCL9-like; TCF/LEF,
T-cell factor/lymphoid enhancer factor; CBP, cAMP response
element-binding protein; PPI, protein−protein interaction;
RCM, ring-closing olefin metathesis; BPB, (S)-2-[N,(N′-
benzylprolyl)amino]benzophenone; Paa, propargylalanine;
Pra, L-propargylglycine; PTHrP, parathyroid hormone-related
peptide; CD, circular dichroism; FP, fluorescence polarization
(14) (a) Belokon, Y. N.; Bespalova, N. B.; Churkina, T. D.; Cisarova,
I.; Ezernitskaya, M. G.; Harutyunyan, S. R.; Hrdina, R.; Kagan, H. B.;
Kocovsky, P.; Kochetkov, K. A.; Larionov, O. V.; Lyssenko, K. A.;
North, M.; Polasek, M.; Peregudov, A. S.; Prisyazhnyuk, V. V.;
Vyskocil, S. Synthesis of alpha-amino acids via asymmetric phase
transfer-catalyzed alkylation of achiral nickel(II) complexes of glycine-
derived Schiff bases. J. Am. Chem. Soc. 2003, 125, 12860−12871.
(b) Belokon, Y. N.; Tararov, V. I.; Maleev, V. I.; Savel’eva, T. F.;
Ryzhov, M. G. Improved procedures for the synthesis of (S)-2-[N-(N′-
benzylprolyl)amino]benzophenone (BPB) and Ni(II) complexes of
Schiff’s bases derived from BPB and amino acids. Tetrahedron:
Asymmetry 1998, 9, 4249−4252.
REFERENCES
■
(1) Polakis, P. Wnt signaling and cancer. Genes Dev. 2000, 14, 1837−
1851.
(2) Takemaru, K. I.; Ohmitsu, M.; Li, F. Q. An oncogenic hub: beta-
catenin as a molecular target for cancer therapeutics. Handb. Exp.
Pharmacol. 2008, 186, 261−284.
(3) Sampietro, J.; Dahlberg, C. L.; Cho, U. S.; Hinds, T. R.;
Kimelman, D.; Xu, W. Q. Crystal structure of a beta-catenin/BCL9/
Tcf4 complex. Mol. Cell 2006, 24, 293−300.
(15) Rohl, C. A.; Chakrabartty, A.; Baldwin, R. L. Helix propagation
and N-cap propensities of the amino acids measured in alanine-based
peptides in 40 volume percent trifluoroethanol. Protein Sci. 1996, 5,
2623−37.
(16) (a) Chapman, R. N.; Dimartino, G.; Arora, P. S. A highly stable
short alpha-helix constrained by a main-chain hydrogen-bond
surrogate. J. Am. Chem. Soc. 2004, 126, 12252−12253. (b) Nikolov-
ska-Coleska, Z.; Wang, R. X.; Fang, X. L.; Pan, H. G.; Tomita, Y.; Li,
P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. M.
Development and optimization of a binding assay for the XIAP BIR3
domain using fluorescence polarization. Anal. Biochem. 2004, 332,
261−273.
(17) (a) Wang, D.; Chen, K.; Kulp Iii, J. L.; Arora, P. S. Evaluation of
biologically relevant short alpha-helices stabilized by a main-chain
hydrogen-bond surrogate. J. Am. Chem. Soc. 2006, 128, 9248−9256.
(b) Shepherd, N. E.; Hoang, H. N.; Abbenante, G.; Fairlie, D. P. Single
turn peptide alpha helices with exceptional stability in water. J. Am.
(4) (a) Haridas, V. From Peptides to Non-Peptide Alpha-Helix
Inducers and Mimetics. Eur. J. Org. Chem. 2009, 30, 5112−5128.
(b) Henchey, L. K.; Jochim, A. L.; Arora, P. S. Contemporary
strategies for the stabilization of peptides in the alpha-helical
conformation. Curr. Opin. Chem. Biol. 2008, 12, 692−697.
(5) (a) Blackwell, H. E.; Grubbs, R. H. Highly efficient synthesis of
covalently cross-linked peptide helices by ring-closing metathesis.
Angew. Chem., Int. Ed. 1998, 37, 3281−3284. (b) Schafmeister, C. E.;
Po, J.; Verdine, G. L. An all-hydrocarbon cross-linking system for
enhancing the helicity and metabolic stability of peptides. J. Am. Chem.
Soc. 2000, 122, 5891−5892.
(6) (a) Bernal, F.; Tyler, A. F.; Korsmeyer, S. J.; Walensky, L. D.;
Verdine, G. L. Reactivation of the p53 tumor suppressor pathway
by a stapled p53 peptide. J. Am. Chem. Soc. 2007, 129, 2456−2457.
́
Chem. Soc. 2005, 127, 2974−2983. (c) Lujan-Upton, H.; Okamoto, Y.;
1145
dx.doi.org/10.1021/jm201125d | J. Med. Chem. 2012, 55, 1137−1146

Journal of Medicinal Chemistry
Article
Walser, A. D. Fluorescence lifetime of terbium(III) as a probe for the
ion-binding properties of tactic poly(methacrylic acids). J. Polym. Sci.,
Part A: Polym. Chem. 1997, 35, 393−398.
(18) Rohl, C. A.; Baldwin, R. L. Deciphering rules of helix stability in
peptides. Methods Enzymol. 1998, 295, 1−26.
1146
dx.doi.org/10.1021/jm201125d | J. Med. Chem. 2012, 55, 1137−1146