Bridged α-helix mimetic small molecules

Article abstract of DOI:10.1039/c9cc03627j

Herein, we report a strategy for generating conformationally restricted α-helix mimetic small molecules by introducing covalent bridges that limit rotation about the central axis of α-helix mimetics. We demonstrate that the bridged α-helix mimetics have enhanced binding affinity and specificity to the target protein due to the restricted conformation as well as extra interaction of the bridge with the protein surface.

Full text of DOI:10.1039/c9cc03627j

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Bridged a-helix mimetic small molecules†
Yeongju Lee,‡a Haeri Im,‡b Sanket Das,a Misook Oh,a Ji Hoon Lee,c Sihyun Ham*b
Cite this: Chem. Commun., 2019,
55, 13311
a
and Hyun-Suk Lim
*
Received 10th May 2019,
Accepted 10th October 2019
DOI: 10.1039/c9cc03627j
rsc.li/chemcomm
Herein, we report
a
strategy for generating conformationally small-molecules,16–31 but many of the current a-helix mimetics
restricted a-helix mimetic small molecules by introducing covalent have unfavourable issues such as low aqueous solubility, syn-
bridges that limit rotation about the central axis of a-helix mimetics. thetic difficulty, structural flexibility, etc.
We demonstrate that the bridged a-helix mimetics have enhanced
In the search for novel a-helix mimetic small molecules, we
binding affinity and specificity to the target protein due to the recently developed a-helix mimetics based on a triazine–piperazine–
restricted conformation as well as extra interaction of the bridge with triazine structure.26 This scaffold has three functional groups
the protein surface.
(R1, R2, and R3) that can match the spatial arrangement of
critical side chain residues at i, i + 3 or i + 4, and i + 7 positions
a-Helices represent the most common protein secondary struc- located on a-helices (Fig. 1B), enabling it to mimic a-helical
tures and play a pivotal role as recognition motifs in many structures and to act as an inhibitor of a-helix-mediated PPIs.
therapeutically relevant protein–protein interaction (PPI) inter- We synthesized a combinatorial one-bead one-compound library
faces. Therefore, the disruption of aberrant PPIs implicated in a of B1500 triazine–piperazine–triazine compounds substituted
wide range of disease states using a-helix mimetic molecules has with various side chains on R1, R2, and R3. Affinity-based
been emerging as an attractive therapeutic strategy.1–5 One such on-bead screening of this library against Mcl-1, an anti-
approach is the introduction of covalent bridges into a-helical apoptotic Bcl-2 family protein, identified 9c as an inhibitor of
peptides that can keep the peptides in stabilized helical confor- the interaction between the Mcl-1 and BH3 protein family such
mations, leading to enhanced binding affinity as well as improved as BAK and BAX. The compound was shown to induce apoptotic
cell permeability and proteolytic stability.6–10 As an alternative to
these peptide-based approaches, Hamilton and co-workers have
pioneered a small-molecule-based strategy.11,12 They developed
small-molecule a-helix mimetics in which non-peptidic small-
molecule scaffolds are substituted with appropriate side chains
to recapitulate the spatial orientation of critical side chains at
i, i + 3 or i + 4, and i + 7 positions on a-helical peptides (Fig. 1A).
The conformation of substituted side chains that match the
spatial orientation is essential for binding to proteins.13–15
A number of efforts have been devoted to develop such
a Department of Chemistry and Division of Advanced Material Science, Pohang
University of Science and Technology (POSTECH), Pohang 37673, South Korea.
E-mail: hslim@postech.ac.kr; Fax: +82-54-279-3399; Tel: +82-54-279-2131
b Department of Chemistry, Sookmyung Women’s University, Seoul 04310,
South Korea. E-mail: sihyun@sookmyung.ac.kr; Fax: +82-2-2077-7321;
Tel: +82-2-710-9410
c New Drug Development Centre, Daegu Gyeongbuk Medical Innovation Foundation,
Fig. 1 Design of bridged a-helix mimetic small-molecules. (A) Structure
of an a-helical peptide with i, i + 4, and i + 7 side-chain positions,
(B) triazine–piperazine–triazine-based a-helix mimetics, (C) structure of
9c, an Mcl-1 inhibitor, having two rotatable bonds, and (D) bridged a-helix
mimetics.
Daegu 41061, South Korea
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc03627j
‡ These authors contributed equally.
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cell death in cancers by disrupting the Mcl-1/BH3 interaction.
Although we have demonstrated that the designed small mole-
cules not only have excellent aqueous solubility and synthetic
accessibility but also are able to serve as a-helix mimetics, their
binding affinity was somewhat weak (Ki = 9.3 mM). The triazine–
piperazine–triazine scaffold can adopt numerous conforma-
tions due to the presence of two rotatable bonds between
piperazine and two triazine rings (Fig. 1C). This inherent
conformational flexibility may cause major entropy loss upon
binding of the scaffold to proteins, thereby resulting in low
binding affinity to its target. Here we report a strategy for
generating conformationally restricted a-helix mimetic small
molecules by limiting rotation about the central axis of a-helix
mimetics.
Inspired by previous studies on the conformational restric-
tion of a-helix mimetics,7,10,32–34 we hypothesized that if the
rotational freedom of the triazine–piperazine–triazine back-
bone is restricted by incorporating a bridge (Fig. 1D), the
binding activity of the scaffold could be improved. To examine
this, we designed a conformationally restricted triazine–piper-
azine–triazine structure, in which the R4 and R5 positions of 9c
are connected by various bridges, such as hydrocarbon, poly-
ethyleneglycol (PEG), aromatic ring- or triazole-containing, and
amide-containing linkers (Table 1). In our previous work, a
docking model of 9c bound by Mcl-1 suggested that three side
chain residues of 9c occupied the BH3 binding pocket of Mcl-1,
Scheme 1 Solid-phase synthesis of bridged triazine–piperazine–triazine
structures (8a–g).
whereas R4 and R5 positions are exposed outside without direct to Mcl-1 protein. Furthermore, appropriate bridges could restrict
interaction with Mcl-1.26 Thus, the introduction of a bridge at the rotation about the central axis of 9c’s triazine–piperazine–
these solvent-exposed sites may not affect the interaction of 9c triazine scaffold and thus increase its binding affinity.
For the synthesis of the designed molecules, we developed a
convenient solid-phase synthetic route (Scheme 1). Initially, the
mono-Alloc-protected amines 1a–g were loaded on PAL alde-
hyde resin by the reductive amination reaction, providing
secondary amines 2a–g. Next, mono-substituted dichlorotria-
zine was coupled to the resin-bound amines 2a–g, and the
chloride on 3a–g was replaced with the nosyl-protected piper-
azine derivative. After deprotection of the nosyl group, cyanuric
chloride was coupled to the resin-bound piperazines 5a–g.
Following the removal of the Alloc group on 6a–g, on-resin
cyclization using DIPEA gave bridged triazine–piperazine–
triazine structures 7a–g. A subsequent amination reaction with
tyramine followed by the cleavage reaction with trifluoroacetic
acid (TFA) afforded the final products 8a–g. The cleaved products
were purified by reverse-phase HPLC. For the preparation of the
compounds with different bridges, the synthetic scheme was slightly
modified (for more details, see Scheme S1 and Table S1, ESI†).
To examine whether the introduction of covalent bridges can
lead to increased conformational rigidity of a-helix mimetics, thereby
improving their binding affinity, we employed a competitive
fluorescence polarization (FP) assay using purified recombinant
Mcl-1 protein and a fluorescently labeled BH3 peptide (KALETL
RRVGDGVQRNHETAF).35 Among the tested compounds, 8f and
8g possessing PEG bridges displayed more than 6-fold improve-
ment in their binding affinities (Ki = 2.6 mM and 2.9 mM,
respectively) compared to the parent compound 9c (Fig. 2).
The enhanced binding activity of the PEG bridged compounds
Table 1 Chemical structures of designed compounds
Chemical structure
of bridge
Chemical structure
of bridge
Compd
Compd
8h
8a–e
8i
8j
8k
8I
8f–g
8m
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Fig. 3 Simulated structures. (A) 9c, (B) PEG-bridged compound 8f (red
coloured in the PEG bridge is oxygen), (C) hydrocarbon-bridged com-
pound 8d, (D) 9c/Mcl-1 complex, (E) 8f/Mcl-1 complex, and (F) 8d/Mcl-1
complex. Yellow and orange colours indicate the interaction between the
molecules and Mcl-1.
Fig. 2 Inhibition curves of 8a–m and 9c for fluorescein-labeled BH3
peptide binding to recombinant Mcl-1 (amino acids 172–320) as deter-
mined by competitive FP assays. Error bars represent standard deviation
from three independent experiments.
forms additional electrostatic interactions with the Mcl-1 surface
(the region coloured in orange in Fig. 3E), the hydrocarbon
bridge in 8d does not provide such additional contacts (Fig. 3F).
This may be because the purely hydrophobic bridge parts are
(8f and 8g) might be attributed to the restricted rotation of the located close to the hydrophilic surface residues (N81, G83, and R84)
triazine–piperazine–triazine axis. In contrast, the compounds of Mcl-1.
bridged by various hydrocarbon linkers showed even decreased
Based on these results, we selected two PEG-bridged com-
or no binding affinity. For compounds 8a–c containing rela- pounds (8f and 8g) as the best inhibitors for further studies.
tively short bridges, ring strain might bend the triazine–piper- Before starting cellular assays, we evaluated their aqueous
azine–triazine structure, causing changes in the spatial location solubility by calculating the partition coefficient. As shown
of three side chains and thereby deteriorating the binding in Table S2 (ESI†), the c log P values of 8f and 8g were 2.38
affinity. To our surprise, however, compounds 8d–e were also and 2.05, respectively, suggesting that they have a desirable
found to have no binding affinity although their bridges have drug-like property in terms of their hydrophilicity. We then
the same or similar length to that of PEG bridged compound 8f. measured their solubility in water. In good agreement with the
One explanation that might account for this observation is that c log P prediction, both compounds exhibited good solubility in
hydrocarbon bridges on 8d–e may interfere with their binding water (4100 mg mLÀ1), which is above the desirable aqueous
to the target protein. The hydrophobicity of all-hydrocarbon solubility for favourable oral biopharmaceutical properties.38
bridges may be unfavourable for protein binding due to the
To assess the ability of the compounds to inhibit the Mcl-1/BAK
hydrophilic nature of the protein surface. Likewise, compounds interaction in living cells, we performed coimmunoprecipitation
8h–m with the other bridges including aromatic ring or experiments. Jurkat T lymphocyte cells were treated with DMSO or
triazole-containing bridges did not exhibit improved binding increasing concentrations of 8f or 8g. The effect of 8f and 8g on the
affinities presumably due to the hydrophobic nature of their Mcl-1/BAK interaction in cells was analysed by Mcl-1 immuno-
bridges (Fig. 2). Indeed, stabilization of helical peptides by all- precipitation and BAK Western blot (Fig. 4A and Fig. S6, ESI†). In
hydrocarbon bridges often compromises the binding affinities the cells treated with 8f or 8g, immunoprecipitated BAK protein was
in spite of their increased helical propensity.10,36,37
significantly decreased in a dose-dependent manner. This clearly
To investigate the impact of covalent bridges and their demonstrates that 8f and 8g are not only cell-permeable, but also
chemical nature on the binding activity, we conducted computa- able to disrupt the Mcl-1 interaction with BAK in cells.
tional studies on the parent (9c), PEG-bridged (8f), and
Because Mcl-1 blocks apoptosis by sequestering the pro-
hydrocarbon-bridged (8d) compounds (Fig. 3). We first performed apoptotic proteins such as BAK, disrupting the Mcl-1/BH3
flexible docking simulations of these compounds to the Mcl-1 interaction by its inhibitors would lead to apoptosis in cancer
surface and then carried out fully atomistic, explicit-water mole- cells. To test this, Jurkat T lymphocyte cells were treated with
cular dynamics simulations to examine the stability of the docked varying concentrations of 8f, 8g, or 9c, and caspase activity was
complexes (ESI†). We found that the binding positions of these measured to monitor apoptosis (Fig. 4B). Not surprisingly, 8f
compounds are similar to that of the BH3 protein (the surface and 8g were far more effective at triggering apoptosis than 9c.
region coloured in yellow in Fig. 3D–F; see also Fig. S4, ESI†). The EC50 values of 8f and 8g are in the low micromolar ranges,
However, a contrasting behavior was observed for the bridge parts which are consistent with their in vitro binding affinities (Fig. 4B).
depending on their chemical nature; while the PEG bridge in 8f To validate the apoptotic effect of 8f and 8g, we used flow
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3 A. J. Wilson, Chem. Soc. Rev., 2009, 38, 3289.
4 V. Azzarito, K. Long, N. S. Murphy and A. J. Wilson, Nat. Chem.,
2013, 5, 161.
5 M. K. P. Jayatunga, S. Thompson and A. D. Hamilton, Bioorg. Med.
Chem. Lett., 2014, 24, 717.
6 L. D. Walensky and G. H. Bird, J. Med. Chem., 2014, 57, 6275.
7 Y. H. Lau, P. de Andrade, Y. Wu and D. R. Spring, Chem. Soc. Rev.,
2015, 44, 91.
8 L. D. Walensky, A. L. Kung, I. Escher, T. J. Malia, S. Barbuto,
R. D. Wright, G. Wagner, G. L. Verdine and S. J. Korsmeyer, Science,
2004, 305, 1466.
9 A. Patgiri, A. L. Jochim and P. S. Arora, Acc. Chem. Res., 2008, 41, 1289.
10 Y. Lee, H. Yoon, S.-M. Hwang, M.-K. Shin, J. H. Lee, M. Oh, S.-H. Im,
J. Song and H.-S. Lim, J. Am. Chem. Soc., 2017, 139, 16056.
11 C. G. Cummings and A. D. Hamilton, Curr. Opin. Chem. Biol., 2010,
14, 341.
12 B. P. Orner, J. T. Ernst and A. D. Hamilton, J. Am. Chem. Soc., 2001,
123, 5382.
13 J. Taechalertpaisarn, R.-L. Lyu, M. Arancillo, C.-M. Lin, L. M. Perez,
T. R. Ioerger and K. Burgess, Org. Biomol. Chem., 2019, 17, 3267.
14 D. Xin, E. Ko, L. M. Perez, T. R. Ioerger and K. Burgess, Org. Biomol.
Chem., 2013, 11, 7789.
15 V. Azzarito, P. Prabhakaran, A. I. Bartlett, N. S. Murphy, M. J. Hardie,
C. A. Kilner, T. A. Edwards, S. L. Warriner and A. J. Wilson, Org.
Biomol. Chem., 2012, 10, 6469.
16 H. Moon and H.-S. Lim, Curr. Opin. Chem. Biol., 2015, 24, 38.
17 F. Lu, S.-W. Chi, D.-H. Kim, K.-H. Han, I. D. Kuntz and R. K. Guy,
J. Comb. Chem., 2006, 8, 315.
Fig. 4 Cellular activities of 8f and 8g on Jurkat T lymphocyte cells. (A) Co-
immunoprecipitation experiments with 8f. The results are representative of
three independent experiments. (B) Caspase 3/7 activity assays. Error bars
represent standard deviations from three independent experiments.
cytometry. As seen in Fig. S7 (ESI†), 8f and 8g exhibited a dose-
dependent increase of cells undergoing apoptosis but not necrosis
whereas the treatment of 9c resulted in increases of both apoptotic
and necrotic cells, indicating the specificity of 8f and 8g. To further
assess the selectivity of 8f and 8g for Mcl-1, we tested whether
these compounds could inhibit the other PPIs that are also mediated
by a-helices, such as Bcl-xL/BH3 and NCOA1/STAT6 interactions
(Fig. S8, ESI†). Although these proteins have similar binding pockets
to accommodate a-helical peptides, 8f and 8g had no inhibitory
effect on both PPIs, demonstrating the selectivity of both com-
pounds for Mcl-1 over the other proteins.
18 A. Volonterio, L. Moisan and J. Rebek, Org. Lett., 2007, 9, 3733.
¨
19 P. Maity and B. Konig, Org. Lett., 2008, 10, 1473.
20 A. Shaginian, L. R. Whitby, S. Hong, I. Hwang, B. Farooqi,
M. Searcey, J. Chen, P. K. Vogt and D. L. Boger, J. Am. Chem. Soc.,
2009, 131, 5564.
21 S. Marimganti, M. N. Cheemala and J.-M. Ahn, Org. Lett., 2009,
11, 4418.
22 J. H. Lee, Q. Zhang, S. Jo, S. C. Chai, M. Oh, W. Im, H. Lu and
H.-S. Lim, J. Am. Chem. Soc., 2011, 133, 676.
23 J. H. Lee and H.-S. Lim, Org. Biomol. Chem., 2012, 10, 4229.
24 J. L. Yap, X. Cao, K. Vanommeslaeghe, K.-Y. Jung, C. Peddaboina,
P. T. Wilder, A. Nan, A. D. MacKerell, W. R. Smythe and S. Fletcher,
Org. Biomol. Chem., 2012, 10, 2928.
25 R. M. Brady, E. Hatzis, T. Connor, I. P. Street, J. B. Baell and
G. Lessene, Org. Biomol. Chem., 2012, 10, 5230.
In summary, we have developed a strategy for creating con-
formationally restricted a-helix mimetic small molecules by intro-
ducing a covalent bridge, thereby limiting rotation about the
central axis of a-helix mimetics. We have demonstrated that the
bridged a-helix mimetics have substantially enhanced binding
affinity and specificity to the target protein likely due to the
restricted conformation as well as extra interaction of the bridge
with the protein surface. Note that our bridged a-helix mimetics
exhibit excellent cell-permeability and aqueous solubility. We
believe that our strategy will be a highly useful means of generat-
ing potent and selective a-helix mimetics as PPI inhibitors.
This work was supported by the National Research Founda-
tion of Korea (NRF) (No. NRF-2017R1A2B3010053 and NRF-
2017M3A9F6029753).
26 M. Oh, J. H. Lee, W. Wang, H. S. Lee, W. S. Lee, C. Burlak, W. Im,
Q. Q. Hoang and H.-S. Lim, Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 11007.
27 A. Barnard, K. Long, H. L. Martin, J. A. Miles, T. A. Edwards,
D. C. Tomlinson, A. Macdonald and A. J. Wilson, Angew. Chem.,
Int. Ed., 2015, 54, 2960.
28 K.-Y. Jung, H. Wang, P. Teriete, J. L. Yap, L. Chen, M. E. Lanning,
A. Hu, L. J. Lambert, T. Holien, A. Sundan, N. D. P. Cosford,
E. V. Prochownik and S. Fletcher, J. Med. Chem., 2015, 58, 3002.
29 J. H. Lee, M. Oh, H. S. Kim, H. Lee, W. Im and H.-S. Lim, ACS Comb.
Sci., 2016, 18, 36.
30 G. V. Raj, G. R. Sareddy, S. Ma, T.-K. Lee, S. Viswanadhapalli, R. Li,
X. Liu, S. Murakami, C.-C. Chen, W.-R. Lee, M. Mann, S. R.
Krishnan, B. Manandhar, V. K. Gonugunta, D. Strand, R. R.
Tekmal, J.-M. Ahn and R. K. Vadlamudi, eLife, 2017, 6, e26857.
31 Z. Hegedus, C. M. Grison, J. A. Miles, S. Rodriguez-Marin, S. L.
Warriner, M. E. Webb and A. J. Wilson, Chem. Sci., 2019, 10, 3956.
32 S. Thompson and A. D. Hamilton, Org. Biomol. Chem., 2012, 10, 5780.
33 K.-Y. Jung, K. Vanommeslaeghe, M. E. Lanning, J. L. Yap, C. Gordon,
P. T. Wilder, A. D. MacKerell and S. Fletcher, Org. Lett., 2013,
15, 3234.
34 I. Arrata, C. M. Grison, H. M. Coubrough, P. Prabhakaran,
M. A. Little, D. C. Tomlinson, M. E. Webb and A. J. Wilson, Org.
Biomol. Chem., 2019, 17, 3861.
35 M. L. Stewart, E. Fire, A. E. Keating and L. D. Walensky, Nat. Chem.
Biol., 2010, 6, 595.
Conflicts of interest
There are no conflicts to declare.
36 G. H. Bird, E. Mazzola, K. Opoku-Nsiah, M. A. Lammert, M. Godes,
D. S. Neuberg and L. D. Walensky, Nat. Chem. Biol., 2016, 12, 845.
Notes and references
1 L. K. Henchey, A. L. Jochim and P. S. Arora, Curr. Opin. Chem. Biol., 37 T. Okamoto, K. Zobel, A. Fedorova, C. Quan, H. Yang,
2008, 12, 692.
W. J. Fairbrother, D. C. S. Huang, B. J. Smith, K. Deshayes and
P. E. Czabotar, ACS Chem. Biol., 2013, 8, 297.
38 C. E. Mu¨ller, Chem. Biodiversity, 2009, 6, 2071.
2 B. N. Bullock, A. L. Jochim and P. S. Arora, J. Am. Chem. Soc., 2011,
133, 14220.
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