α-Conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure

Article abstract of DOI:10.1021/ja206408q

The two disulfide bonds of α-conotoxin ImI, a peptide antagonist of the α7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a γ-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to α-conotoxin ImI by ¹H NMR. The antagonistic activity at the α7 nAChR of cystathionine analogue 3 (pIC₅₀ = 6.41 ± 0.09) was identical to that of α-conotoxin ImI (1, pIC₅₀ = 6.41 ± 0.09), whereas those of 2 (pIC₅₀ = 5.96 ± 0.09) and 4 (pIC ₅₀ = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S=O) to a 3-fold increase (3S=O_B) in potencies.

Full text of DOI:10.1021/ja206408q

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α-Conotoxin ImI Incorporating Stable Cystathionine Bridges
Maintains Full Potency and Identical Three-Dimensional Structure
Zoltan Dekan, Irina Vetter, Norelle L. Daly, David J. Craik, Richard J. Lewis, and Paul F. Alewood*
Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Queensland, Australia
S Supporting Information
b
ABSTRACT: The two disulfide bonds of α-conotoxin ImI,
a peptide antagonist of the α7 nicotinic acetylcholine
receptor (nAChR), were systematically replaced with isos-
teric redox-stable cystathionine thioethers. Regioselective
thioether formation was accomplished on solid support
through substitution of a γ-chlorohomoalanine by an in-
tramolecular cysteine thiol to produce hybrid thioether/
Figure 1. Cystathionine analogues of α-ImI.
disulfide analogues (2 and 3) as well as a dual cystathionine
analogue (4) that were found to be structurally homologous
to α-conotoxin ImI by 1H NMR. The antagonistic activity at
the α7 nAChR of cystathionine analogue 3 (pIC50 = 6.41 (
Common synthetic approaches for thioether cyclization of
peptides include thiol addition to an alkene, substitution of a
leaving group by a thiol, desulfurization of a disulfide, and
cyclization through amide bonds using preformed orthogon-
ally protected thioether building blocks.19 While each of these
methods has its own advantage, sequence-independent stereo-
and regioselectivity as well as simplicity of synthesis are desired
for general application to a wider range of peptides, especially
where multiple Ctt's are required.
This study focuses on the synthesis and characterization of the
potent and selective α-conotoxin ImI (α-ImI)24 incorporating either
one or two cystine bond thioether replacements. It was anticipated
that such replacements would generate a mimetic stable to reduc-
tionwhile maintaining structure and function identical to those of
the native molecule. An Fmoc solid-phase Ctt synthesis strategy
was undertaken via the intramolecular alkylation of a selectively
deprotected cysteine thiol by γ-chlorohomoalanine (ClhAla)25
on the growing protected peptide chain to incorporate regios-
pecifically either one or two Ctt moieties.
α-ImI was chosen as a model bioactive peptide for the synthesis
of Ctt analogues 2, 3, and 4 (Figure 1), as the two disulfide bonds
are largely buried and disulfide bond replacements must be finely
tuned if full structural and functional integrity is to be main-
tained. α-ImI is a selective peptide antagonist of α7 nicotinic
acetylcholine receptors (nAChR) with the sequence of GCCSD-
PRCAWRC-NH2, containing two disulfide bonds with the con-
nectivity Cys 1À3, Cys 2À4. α-ImI is a well-characterized molecule
that provides an ideal model for the evaluation of the effects of
disulfide bond mimics on structure and function, as it allows a
direct comparison of Ctt replacements withitspreviouslyreported
diselenide7 and dicarba18 counterparts.
Cystathionine-containing α-ImI analogues 2À4 were pre-
pared as outlined in Scheme 1. The linear precursors 5 were syn-
thesized on-resin using standard Fmoc solid-phase peptide syn-
thesis (SPPS) followed by conversion of Hse(TBDMS) to ClhAla
and deprotection of Cys(StBu) [where Hse is homoserine,
0.09) was identical to that of α-conotoxin ImI (1, pIC50
=
6.41 ( 0.09), whereas those of 2 (pIC50 = 5.96 ( 0.09) and
4 (pIC50 = 5.89 ( 0.09) showed a modest decrease. The
effect of oxidation of the thioethers to sulfoxides was also
investigated, with significant changes in the biological
activities observed ranging from a >30-fold reduction
(2SdO) to a 3-fold increase (3SdOB) in potencies.
isulfide bonds are a dominant structural feature of many
D
peptides and proteins that are essential for maintaining well-
defined three-dimensional structures that allow high-affinity
interactions with specific molecular targets.1 However, many
disulfide bonds are susceptible to reduction and scrambling
in vivo by glutathione2 as well as redox enzymes,3 potentially
resulting in the loss of the desired biological activity of the
peptide. A number of alternative linkages with increased redox
stabilities including lactams,4 diselenides,5À10 thioethers,11À16
and dicarba17,18 analogues have previously been introduced into
biologically active peptides with some success in maintaining
structure and activity.
Lanthionine thioethers, in which one of the sulfur atoms
of the disulfide bond is removed, have been assessed
extensively19 as disulfide bond replacements or as synthetic
structural constraints, and are also found in some naturally
occurring bioactive peptides such as the lantibiotics.20,21
Although lanthionines make up an interesting class of com-
pounds and have previously been used successfully as syn-
thetic disulfide bond replacements, they are not isosteric
with cystine. The cystathionine (Ctt) thioether, where one
of the sulfur atoms of a disulfide bond is substituted with a
methylene group, should more closely approximate the geo-
metry of cystine than dicarba or lanthionine analogues and is
therefore hypothesized to cause the minimum of structural
perturbations.16,22,23
Received: July 11, 2011
Published: September 07, 2011
r
2011 American Chemical Society
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Scheme 1. Solid-Phase Synthesis of Cystathionine Analogues of α-ImI, 4 and 3a
a Reagents and conditions: (a) 10 equiv of Ph3PCl2, DCM, 6 h; (b) 100 equiv of Bu3P in 5% H2O/THF, 2 h; (c) 0.1 M NaHCO3 in DMF/H2O (3:2),
72 h; (d) SPPS: Fmoc-Hse(TBDMS)-OH; (e) SPPS: Boc-Gly-OH; (f) 3% TFA/5% TIPS in DCM, 10 Â 3 min; (g) SPPS: Fmoc-Cys(Trt)-OH; (h)
95% TFA/2.5% TIPS/2.5% H2O, 2 h; (i) 0.1 M NH4HCO3 (aq), pH 8.2, 6 h; (j) 0.1% H2O2/0.1% TFA (aq), 35°C, 16 h. * 2 was prepared analogously
to 3 by exchanging the positions of Cys 2(Trt) and Hse 3(TBDMS), and those of Cys 8(Trt) and Cys 12(StBu)).
TBDMS is tert-butyldimethylsilyl, and StBu is tert-butylthio],
which proceeded smoothly irrespective of the type of solid
support used (polystyrene or Tentagel-S). Following 6 and 2 h
treatments with Ph3PCl2 and Bu3P, respectively, the desired pro-
duct containing the Cl-hAla and the S-deprotected cysteine 6 was
obtained. To optimize the on-resin intramolecular thiol alkyla-
tion, conditions were evaluated by varying the solvent (DMF or
DMF/H2O), the base (DIEA, NMM, Na2CO3, or NaHCO3)
and the type of solid support (polystyrene or Tentagel-S). Initial
attempts at intramolecular alkylation of Cys 12 with ClhAla 3
(to form the 2À4 Ctt, 7) on polystyrene solid support using pre-
viously reported conditions15,26,27 with DMF as the solvent and
either DIEA or NMM as the base did not result in the formation
of the desired Ctt product. Attempts at using the same conditions
on Tentagel-S resin also failed to produce a thioether bond, as
only the linear precursors were recovered in each case. The use of
Na2CO325 in DMF/H2O gave no 7, though thioether cyclization
was eventually accomplished by treatment of 6 with 0.1 M
NaHCO3 in DMF/H2O (3:2) on Tentagel-S resin. The same
conditions were used successfully with subsequent substitution
reactions to produce the dual Ctt analogue 4, following the
coupling of the second Hse(TBDMS) and final Gly residues,
O(TBDMS) to -Cl conversion, and Cys(Trt) deprotection.
Tryptic digest of the purified final product showed a single
HPLC peak containing the mass of the cyclized Ctt peptide +
36 Da (Supporting Information Figure S2), corresponding to
cleavage at both Arg residues with the fragments being held
together by the two thioether bonds, confirming that both
cyclizations had indeed taken place. The hybrid cystine/Ctt
analogues 2 and 3 were prepared by replacing Hse 3 and Hse 2
with a Cys(Trt) residue respectively, and following the forma-
tion of one thioether bridge, completion of chain assembly 9,
cleavage from the resin, and formation of the disulfide bond by
air oxidation of the crude peptides in aqueous buffer.
Although not observed during the synthesis and handling of
these particular peptides, thioethers can be susceptible to the for-
mation of sulfoxides, which may cause further structural changes
to the molecule. To investigate these effects on biological activity,
sulfoxide analogues of 2 and 3 were prepared by oxidation of the
thioether bridge using H2O2.16 Interestingly, the oxidation of 2
produced only a single product (2SdO, as determined by RP-
HPLC and MS) rather than the expected isomeric mixture of
sulfoxides as observed following the oxidation of 3, which were
isolated and designated as 3SdOA (earlier eluting) and 3SdOB
(later eluting).
Functional activities of the Ctt analogues are summarized in
Figure 2. Replacement of the Cys 2À4 disulfide with a Ctt bridge
(3) did not show any difference in activity (pIC50 = 6.41 ( 0.09)
compared to α-ImI (1). The two isomers produced by the
oxidation of the thioether of 3 each displayed different activities,
with 3SdOA showing a 2-fold decrease (pIC50 = 6.10 ( 0.06)
and 3SdOB showing a 3-fold increase (pIC50 = 6.92 ( 0.05) in
potency compared to 1, suggesting that significant structural
changes may be occurring due to the altered geometry of the
bridge.
The substitution of the Cys 1À3 disulfide (2) resulted in a
3-fold reduction in potency (pIC50 = 5.96 ( 0.09), and similarly,
a 3-fold decrease in activity was observed when both the Cys 1À3
and the Cys 2À4 disulfides were replaced (4, pIC50 = 5.89 (
0.09). The Cys 1À3 disulfide bond of α-conotoxins is predicted
to be involved in binding to nAChRs through hydrophobic inter-
actions with an internal vicinal disulfide bond of the receptor.28
Thus, the drop in activity of 2 and 4 may be attributed to the
decreased hydrophobicity of the thioether compared to the
native disulfide (based on retention time, see Supporting Infor-
mation Figure S1) rather than a result of structural changes.
Further reduction in the hydrophobicity of this bridge by oxi-
dation of Ctt 1À3 to its sulfoxide (2SdO) resulted in an even
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COMMUNICATION
Figure 2. (a) Representative concentrationÀresponse curves showing
inhibition of choline-induced Ca2+ responses from SH-SY5Y cells
expressing the human α7 nAChR by the cystathionine analogues of
α-ImI. (b) Comparison of pIC50 values (*IC50 of 2SdO estimated from
extrapolation of the concentrationÀresponse curve). Data are presented
as mean ( SEM of n = 3 replicates and are representative of 3À5
independent experiments.
Figure 3. (a) Differences in 1H NMR Hα shifts of 2À4 relative to 1. (b)
Overlay of the average of the 20 lowest energy structures of 1 (PDB
1IM1) (red), 2 (blue), and 3 (green). Cystine bridges are shown in
yellow; Ctt 1À3 of 2 and Ctt 2À4 of 3 are shown in light blue and light
green, respectively. Yellow balls represent sulfur atoms, and the Cγ of
cystathionines is highlighted in gray.
analysis showed that the Ctt analogues caused minimal perturba-
tions to the native peptide, and the functional assays revealed no
or very modest losses in biological activities, confirming the con-
servative nature of these substitutions and further supporting
their usefulness as stable disulfide bond mimetics. Oxidation of
the thioether bridges to sulfoxides provided fast and convenient
access to a new set of analogues with significantly altered bio-
logical activities and provided further insight into the structureÀ
activity relationships of α-ImI.
greater loss of activity (>30 fold), although structural similarity to
α-ImI was not confirmed in this case. This trend is consistent
with the increase in activities of other α-conotoxins at nAChRs
observed following the replacement of Cys 1À3 with the more
hydrophobic selenocystine.29
Analysis of the thioethers 2À4 by two-dimensional 1H NMR
confirmed the presence of a single conformation for each
analogue. The concordance of the backbone Hα chemical shifts
(Δ Hα < 0.1) for most residues with those of 130 indicates that
both Cys 1À3 and Cys 2À4 cystine-to-cystathionine substitu-
tions were structurally conservative (Figure 3A). The slight
upfield shifts of Ctt 2 Hα of 2 and 4 and Ctt 3 Hα of 3 compared
to those of Cys 2 and Cys 3 of 1, respectively, are consistent with
the increased shielding effect of the introduced methylene group
compared to that of the native sulfur atom. The three-dimen-
sional structures of 2 (BMRB 21007) and 3 (BMRB 21006) were
calculated from NOE and dihedral restraints (see Supporting
Information Table S1). Superposition of the backbone atoms of
2 and 3 with those of 1 (Figure 3B) resulted in low rmsd values of
0.35 and 0.37 for residues 2À10 (0.90 and 0.79 for residues
1À12) between the average structures of 1 and 2 and between
those of 1 and 3, respectively, which confirms a high degree of
structural homology. The differences in the rmsd values for
residues 1À12 compared to those of 2À10 are due to increased
structural disorder at residues 1, 11, and 12 observed in all three
sets of structures, including that of the native molecule.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures and additional figures (S1 and S2). This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
p.alewood@imb.uq.edu.au
’ ACKNOWLEDGMENT
This work was supported by Discovery Grant DP1096866
from the Australian Research Council (P.F.A.) and Program
Grant 569927 (P.F.A., R.J.L.), Research Fellowship 455840
(R.J.L.), and Professorial Fellowship 569603 (D.J.C.) from the
National Health and Medical Research Council of Australia,
Australian Based Biomedical Fellowship 569918 (I.V.), and a
Queensland Smart State Fellowship (N.L.D.). The authors
In summary, each of the disulfide bonds of α-ImI was
successfully replaced with isosteric cystathionines on solid support
using a simple intramolecular Cys alkylation strategy. Structural
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Journal of the American Chemical Society
COMMUNICATION
acknowledge Alun Jones for performing high-resolution mass
spectrometry.
’ REFERENCES
(1) Schulz, G. E.; Schirmer, R. H. Principles of protein structure;
Springer: New York, 1979.
(2) Gilbert, H. F. Methods Enzymol. 1995, 251, 8–28.
(3) Holmgren, A.; Bjornstedt, M. Methods Enzymol. 1995, 252,
199–208.
(4) Hargittai, B.; Sole, N. A.; Groebe, D. R.; Abramson, S. N.;
Barany, G. J. Med. Chem. 2000, 43, 4787–4792.
(5) Walewska, A.; Zhang, M. M.; Skalicky, J. J.; Yoshikami, D.;
Olivera, B. M.; Bulaj, G. Angew. Chem., Int. Ed. 2009, 48, 2221–2224.
(6) Pegoraro, S.; Fiori, S.; Rudolph-Bohner, S.; Watanabe, T. X.;
Moroder, L. J. Mol. Biol. 1998, 284, 779–792.
(7) Armishaw, C. J.; Daly, N. L.; Nevin, S. T.; Adams, D. J.; Craik,
D. J.; Alewood, P. F. J. Biol. Chem. 2006, 281, 14136–14143.
(8) Muttenthaler, M.; Alewood, P. F. J. Pept. Sci. 2008, 14,
1223–1239.
(9) Fiori, S.; Pegoraro, S.; Rudolph-Bohner, S.; Cramer, J.; Moroder,
L. Biopolymers 2000, 53, 550–564.
(10) Moroder, L. J. Pept. Sci. 2005, 11, 187–214.
(11) Bondebjerg, J.; Grunnet, M.; Jespersen, T.; Meldal, M. Chem-
biochem 2003, 4, 186–194.
(12) Galande, A. K.; Bramlett, K. S.; Burris, T. P.; Wittliff, J. L.;
Spatola, A. F. J. Pept. Res. 2004, 63, 297–302.
(13) Rudinger, J.; Jost, K. Experientia 1964, 20, 570–571.
(14) Rew, Y.; Malkmus, S.; Svensson, C.; Yaksh, T. L.; Chung, N. N.;
Schiller, P. W.; Cassel, J. A.; DeHaven, R. N.; Goodman, M. J. Med.
Chem. 2002, 45, 3746–3754.
(15) Mayer, J. P.; Zhang, J. W.; Groeger, S.; Lu, C. F.; Jarosinski,
M. A. J. Pept. Res. 1998, 51, 432–436.
(16) Knerr, P. J.; Tzekou, A.; Ricklin, D.; Qu, H.; Chen, H.; van der
Donk, W. A.; Lambris, J. D. ACS Chem. Biol. 2011, 6, 753–760.
(17) Stymiest, J. L.; Mitchell, B. F.; Wong, S.; Vederas, J. C. Org. Lett.
2003, 5, 47–49.
(18) MacRaild, C. A.; Illesinghe, J.; van Lierop, B. J.; Townsend,
A. L.; Chebib, M.; Livett, B. G.; Robinson, A. J.; Norton, R. S. J. Med.
Chem. 2009, 52, 755–762.
(19) Paul, M.; van der Donk, W. A. Mini-Rev. Org. Chem. 2005,
2, 23–37.
(20) Jung, G. Angew. Chem., Int. Ed. Engl. 1991, 30, 1051–1068.
(21) Willey, J. M.; van der Donk, W. A. Annu. Rev. Microbiol. 2007,
61, 477–501.
(22) Chen, C. S.; Srikrishnan, T.; Parthasarathy, R. Biochim. Biophys.
Acta 1978, 538, 534–540.
(23) Hidaka, Y.; Ohmori, K.; Wada, A.; Ozaki, H.; Ito, H.; Hirayama,
T.; Takeda, Y.; Shimonishi, Y. Biochem. Biophys. Res. Commun. 1991,
176, 958–965.
(24) Mcintosh, J. M.; Yoshikami, D.; Mahe, E.; Nielsen, D. B.; Rivier,
J. E.; Gray, W. R.; Olivera, B. M. J. Biol. Chem. 1994, 269, 16733–16739.
(25) Yu, L.; Lai, Y. H.; Wade, J. V.; Coutts, S. M. Tetrahedron Lett.
1998, 39, 6633–6636.
(26) Mayer, J. P.; Heil, J. R.; Zhang, J. G.; Munson, M. C. Tetrahedron
Lett. 1995, 36, 7387–7390.
(27) Roberts, K. D.; Ede, N. J. J. Pept. Sci. 2007, 13, 811–821.
(28) Ulens, C.; Hogg, R. C.; Celie, P. H.; Bertrand, D.; Tsetlin, V.;
Smit, A. B.; Sixma, T. K. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3615–3620.
(29) Muttenthaler, M.; Nevin, S. T.; Grishin, A. A.; Ngo, S. T.; Choy,
P. T.; Daly, N. L.; Hu, S. H.; Armishaw, C. J.; Wang, C. I. A.; Lewis, R. J.;
Martin, J. L.; Noakes, P. G.; Craik, D. J.; Adams, D. J.; Alewood, P. F.
J. Am. Chem. Soc. 2010, 132, 3514–3522.
(30) Rogers, J. P.; Luginbuhl, P.; Shen, G. S.; McCabe, R. T.;
Stevens, R. C.; Wemmer, D. E. Biochemistry 1999, 38, 3874–3882.
15869
dx.doi.org/10.1021/ja206408q |J. Am. Chem. Soc. 2011, 133, 15866–15869