Preparation of Selenoinsulin as a Long-Lasting Insulin Analogue

Article abstract of DOI:10.1002/anie.201701654

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U^A,C7U^B] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ_1/2≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.

Full text of DOI:10.1002/anie.201701654

Angewandte
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International Edition: DOI: 10.1002/anie.201701654
German Edition: DOI: 10.1002/ange.201701654
Protein Folding
Preparation of Selenoinsulin as a Long-Lasting Insulin Analogue
Kenta Arai+, Toshiki Takei+, Masaki Okumura+, Satoshi Watanabe+, Yuta Amagai,
Yuya Asahina, Luis Moroder, Hironobu Hojo,* Kenji Inaba,* and Michio Iwaoka*
Abstract: Synthetic insulin analogues with a long lifetime are
current drug targets for the therapy of diabetic patients. The
replacement of the interchain disulfide with a diselenide bridge,
which is more resistant to reduction and internal bond rotation,
can enhance the lifetime of insulin in the presence of the
insulin-degrading enzyme (IDE) without impairing the hor-
monal function. The [C7UA,C7UB] variant of bovine pancre-
atic insulin (BPIns) was successfully prepared by using two
selenocysteine peptides (i.e., the C7U analogues of A- and B-
chains, respectively). In a buffer solution at pH 10 they
spontaneously assembled under thermodynamic control to
the correct insulin fold. The selenoinsulin (Se-Ins) exhibited
a bioactivity comparable to that of BPIns. Interestingly,
degradation of Se-Ins with IDE was significantly decelerated
(t1/2 ꢀ 8 h vs. ꢀ 1 h for BPIns). The lifetime enhancement could
be due to both the intrinsic stability of the diselenide bond and
local conformational changes induced by the substitution.
under physiological conditions and slowly releases active
insulin monomers.
In contrast, the insulin-degrading enzyme (IDE) is
a possible alternative target for diabetes therapy. IDE,
which is involved in clearance of insulin and amyloid b
(Ab),[5] is found in the liver and kidneys. Recent research has
revealed that synthetic IDE inhibitors increase circulation of
insulin by preventing its degradation in the liver, thus
resulting in improvement of the postprandial glucose toler-
ance.[6] However, other research suggests that IDE inhibitors
could induce accumulation of Ab in the brain,[7] and would
lead to Ab-mediated cognitive impairment. Hence, the design
of long-lasting insulin analogues resistant against IDE would
be desirable.[8]
In this study, we have attempted a new approach to a long-
lasting insulin analogue by exploiting the unique chemical
properties of a diselenide bond. Namely, introduction of two
juxtaposed selenium atoms to the insulin analogue could lead
to a higher kinetic and thermodynamic stability than that of
the wild-type without affecting the bioactivity. This new
I
nsulin, a small globular protein (5.8 kDa), comprises two
peptide chains, the A-chain (Ins-A, 21 amino acid residues)
and B-chain (Ins-B, 30 amino-acid residues). The native
structure in a monomeric active state is stabilized by two
interchain disulfide bridges, CysA7-CysB7 and CysA20-CysB19, in
strategy is based primarily on the higher rotational barrier of
À1
À
À
a Se Se bond (ca. 4 kcalmol ) than that of an S S bond (ca.
3 kcalmolÀ1),[9] and secondarily on the differing redox poten-
tials of diselenides (E’0 À381 mV) and disulfides (E’0
addition to one intrachain disulfide linkage, CysA6-CysA11 [1]
.
À215 mV),[10] even though the structural features of a Se Se
À
Considerable efforts have been directed toward development
of various insulin analogues[2] which imitate either bolus
secretion of insulin for expeditiously reducing postprandial
blood glucose levels[3] or basal secretion of insulin to control
the glucose level for an entire day.[4] The latter long-acting
analogues have been designed so that insulin forms infusible
precipitates or soluble oligomers (hexamer or dihexamer)
À
and S S bond are very similar to each other.
À
The lower redox potential of a Se Se bond is also
advantageous for preparation of a diselenide insulin analogue
À
(selenoinsulin) because formation of a Se Se bridge should
occur independently of the presence of additional cysteine
residues, and thus facilitate the correct oxidative folding of
cysteine-rich peptides. This advantage was already fully
confirmed by the quantitative oxidative production of the
natural isomer of endothelin[11] and both the natural and non-
natural isomers of apamin.[12] More recently, it was success-
fully used in the generation of the correct natural diselenide/
disulfide isomers of many cysteine-rich peptides (for reviews
see Ref. [13]). The obtained Sec-substituted peptides were
found to retain the native structures with biological activities
comparable to those of the wild-types, thus confirming the
[*] Dr. K. Arai,[+] T. Takei,[+] Prof. Dr. M. Iwaoka
Department of Chemistry, School of Science, Tokai University
Kitakaname, Hiratsuka-shi, Kanagawa 259-1292 (Japan)
E-mail: miwaoka@tokai.ac.jp
T. Takei,[+] Dr. Y. Asahina, Prof. Dr. H. Hojo
Institute for Protein Research, Osaka University
Yamadaoka, Suita-shi, Osaka 565-0871 (Japan)
E-mail: hojo@protein.osaka-u.ac.jp
Dr. M. Okumura,[+] Dr. S. Watanabe,[+] Dr. Y. Amagai, Prof. Dr. K. Inaba
Institute of Multidisciplinary Research for Advanced Materials,
Tohoku University
À
À
isomorphous structural character of the Se Se versus the S S
bridges.[13a,c,14]
By employing bovine pancreatic insulin (BPIns; Fig-
ure 1A) as a model protein, we designed a new selenoinsulin
analogue in which CysA7 and CysB7 are replaced with two
selenocysteine (Sec; U) residues. We chose this strategy not
only because it would bring significant conformational
stabilization around the solvent-exposed CysA7-CysB7 bridge
Aoba-ku, Sendai, 2-1-1 (Japan)
E-mail: kinaba@tagen.tohoku.ac.jp
Prof. Dr. L. Moroder
Max Planck Institute of Biochemistry
Am Klopferspitz 18, 82152 Martinsried (Germany)
[+] These authors contributed equally to this work.
À
by substitution with a more robust Se Se bridge, but also
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201701654.
because it would inhibit undesired formation of the swap
species having an incorrect CysA6-CysB7 bridge.[15] By applying
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folding intermediates present in the reaction solution were
then analyzed by RP-HPLC (Figure 2) and MALDI-TOF-MS
analysis. Various species, that is, 1SeS of 1 (peaks B, C, and D)
and reduced forms of 1 and 2 (peaks A and G), were
Figure 1. Primary amino-acid sequences and disulfide pattern. A) Wild-
type BPIns. B) Synthetic selenoinsulin A-chain (Se-Ins-A; 1) and B-
chain (Se-Ins-B; 2). U and Pys represent a selenocysteine residue and
a 2-pyridylsulfanyl group, respectively.
Figure 2. RP-HPLC chromatograms obtained from oxidative chain
assembly experiments of Se-Ins in a 25 mm sodium bicarbonate buffer
solution (pH 10.0). Folding of Se-Ins was initiated by addition of DTT
under the conditions of [1]=[2]=184 mm, [DTT]=4 mm, and
Fmoc-based solid-phase peptide synthesis (SPPS), two sele-
nopeptides, Se-Ins-A (1) and Se-Ins-B (2; Figure 1B), were
prepared with yields of 9 and 6%, respectively. The Sec
residue was introduced to a growing peptide chain on the
resin by using Fmoc-Sec(MPM)-OH with DIC-HOBt activa-
tion. Since the fully reduced and unprotected form of the
C7UA analogue could not be obtained as a single product,
probably because of facile formation of either the intrachain
[urea]=0.75m at 48C. The symbols indicate. A=fully reduced 1,
A
À
À
B–D=1SeS , E and F=fully oxidized 1 having one S S and one Se S
linkage, G=reduced 2, N=native Se-Ins, H=oxidized 2, I=[Se-Ins-
B]2 =a homodimer of Se-Ins-B, x=not identified products, 1’=Se-Ins-
A-DTT2 conjugate with two SH groups, and 2’=Se-Ins-B-DTT2 con-
jugate.
À
À
Se S or S S linkage at various positions, 1 was isolated as a 2-
À
pyridylsulfanyl (Pys)-protected derivative with one Se S
identified along with oxidized 2 (peak H). A homodimer of
Se-Ins-B (peak I) was also observed in a significant amount.
After 24 hours, most of the species initially observed
converged to the major product (peak N), concomitantly
generating the fully oxidized 1 (peaks E and F) and some
unidentified species (peaks x) as minor products (Figure 2).
The major product was then isolated and characterized as
correctly folded selenoinsulin (Se-Ins) by single-crystal X-ray
analysis (see below). The yield of the isolated product was up
to 21% as determined by amino-acid analysis. By optimizing
the reaction conditions (see Table S1), the yield could be
slightly increased to 24% by use of TCEP, instead of DTT, as
a reductant to initiate the chain-assembly reaction. The yield
was further increased to 27% when the chain assembly was
carried out in the presence of less urea (0.5m). It is worth
noting that this preparation is the first example to show that
the Cys-to-Sec substitution method[10–13] is effective for the
oxidative folding of heterodimeric proteins.
linkage. According to reverse-phase (RP) HPLC analysis
(see Figure S1D in the Suppporting Information), 1 contained
a single component (either of 1a, 1b, or 1c) but the exact
À
position of the Se S linkage could not be determined. The
selenopeptide 2, the C7UB analogue, was obtained as the
À
oxidized form with a Se S linkage. Identities and purities of
1 and 2 were unambiguously confirmed by amino-acid
analysis as well as by MALDI-TOF-MS and HPLC analyses
(see the Supporting Information).
The oxidative chain assembly was initiated by treatment
of the 1:1 mixture of 1 and 2 with DTT (four equivalents with
respect to the sum of Sec and Cys residues), which according
to its redox potential[10] should remove the Pys groups from
À
the Cys residues as well as reduce the Se S linkages between
the Sec and Cys residues, at 48C in a sodium bicarbonate
buffer solution at pH 10.0 containing 0.75m urea to prevent
aggregation of 1. The reduced peptides 1 and 2, with free thiol
(SH) and selenol (SeH) functional groups, were subsequently
allowed to assemble spontaneously under thermodynamic
control by air oxygen. After 1 minute, an aliquot of the
solution was taken, and the reaction was quenched with
aqueous 2-aminoethyl methanethiosulfonate (AEMTS),[16]
which rapidly converted the free SH and SeH groups into
The crystal structure of Se-Ins thus isolated was deter-
mined at 1.45 ꢀ resolution by the single anomalous dispersion
(SAD) method using anomalous scattering of the selenium
atoms. The crystallographic data for Se-Ins are summarized in
Table S2. Superposition of the X-ray crystal structures of Se-
Ins and BPIns (PDB code: 2bn3)[17] demonstrates that Se-Ins
has a nearly identical structure to that of BPins with an
+
+
-SSCH2CH2NH3 and -SeSCH2CH2NH3 , respectively. The
2
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RMSD of 0.985 ꢀ over all identical atoms in both structures
(Figure 3A) though small but significant changes have been
induced by the diselenide bond substitution as described
below. The two Sec residues form a diselenide bridge at the
native position.
Figure 4. Se-Ins or refolded BPIns activates insulin signaling pathway.
Serum-starved HeLa cells were stimulated with 1 mm of either BPIns,
refolded BPIns, or Se-Ins for 2.5 min. A) Whole-cell lysates analyzed by
immunoblotting. B) The quantitative data. Bars are shown as means
ÆSEM (n=3). The signal intensity of phosphorylated Akt and phos-
phorylated GSK3b was normalized against that of Akt and GSK3b,
respectively.
Figure 3. A) Superposition of X-ray crystal structures of Se-Ins (blue;
PDB code: 5azz) and BPIns (gray; PDB code: 2bn3). B) Comparison of
the N-terminal regions of B-chains between BPIns (left panel) and Se-
Ins (right panel). Shorter distances between Phe1 and its adjacent
residues in the B-chain of Se-Ins suggest that the N-terminal half of
the B-chain is more tightly packed in Se-Ins.
Figure 5. Degradation of BPIns and Se-Ins by insulin-degrading
enzyme (IDE). Quantitative analyses of native BPIns (black) and Se-Ins
(red). Values are the mean Æ SD of three independent experiments.
The results of the X-ray analysis suggest that the function
of Se-Ins should be comparable to that of native BPIns. This
function was confirmed by experiments using cultured cells
(Figure 4). Upon stimulation by Se-Ins, the phosphorylation
levels of Akt and GSK3b were significantly increased in HeLa
cells, as by stimulation with BPIns.[18] The data indicate that
Se-Ins, as well as the synthetic folded BPIns, retain the
physiological activity of insulin even after replacement of an
interchain disulfide with a diselenide bridge.
resulting in the enhanced resistance to IDE degradation. The
second scenario could be drawn based on the local stabiliza-
tion around the N- and C-terminal regions of Se-Ins. The
crystal structure of the IDE-Ins complex showed that the N-
and C-terminal regions of each chain are partially unfolded at
the initial step of the degradation by IDE.[19] Local conforma-
À
The rate of degradation by IDE was subsequently
compared for Se-Ins and BPIns (Figure 5). By treating the
insulin samples with IDE at 308C in a Tris buffer solution at
pH 8.0, a number of digested peptide fragments were
produced as revealed by RP-HPLC analysis (see Figure S3).
Of interest, the degradation rate of Se-Ins was about eight
times slower than that of BPIns in terms of the half lifetime
(t1/2 ꢀ 8 h for Se-Ins vs. ꢀ 1 h for BPIns), thus strongly
suggesting a long-lasting nature of Se-Ins in vivo. Though
not fully evidenced, the following scenarios could account for
this observation. Firstly, replacement of the solvent-exposed
CysA7-CysB7 bridge with a diselenide bond, which is more
resistant to reduction and internal bond rotation, should
confer extra structural robustness on the insulin fold, thus
tional changes induced by the Se Se bond substitution
enhances the interactions between the N-terminus (PheB1)
and its neighboring residues in the Se-Ins B-chain (Fig-
ure 3B). We also note that water-mediated interactions
involving ThrB27, IleA2, and TyrA19 are further stabilized at
the interface of the N-terminal region of the A-chain and the
C-terminal region of the B-chain in Se-Ins (see Figure S2).
Such local stabilization found in the N- and C-terminal
regions likely allows Se-Ins to prevent the partial unfolding by
IDE to a significant extent.
In summary, we have applied the Cys-to-Sec substitution
methodology to chain assembly/folding of BPIns and have
succeeded in the synthesis of a new [C7UA,C7UB] variant of
insulin in reasonable yields upon isolation (up to 27%). The
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Acknowledgments
K.A. and M.O. acknowledge Japan Society for the Promotion
of Science (JSPS) for research fellowships for young scientists.
We thank S. Shimodaira for the synthesis of the selenocys-
teine derivative and Dr. K. Mizuno (Tohoku University) for
providing the antibodies. We also thank the beamline
scientists at the Photon Factory for their help in the X-ray
diffraction data collection. This work was supported by
KAKENHI Grant Number 26888016 for Research Activity
Start-up (to K.A.), in part by Research and Study Project of
Tokai University, Educational System General Research
Organization (to K.A. and M.I.), Cooperative Research
Program of Institute for Protein Research, Osaka University
CR-15-01 (to M.I. and H.H.), Cooperative Research Program
of Network Joint Research Center for Materials and Devices
(to M.I. and K.I.), CREST, JST [to K.I. (JPMJCR13M6)],
Grant-in-Aids for Scientific Research on Innovative Areas
from MEXT [to K.I. (26116005) and M.O. (15641922)],
Takeda Science Foundation (to K.I.), Uehara Memorial
Foundation (to M.O.), and the Platform Project for Support-
ing Drug Discovery and Life Science Research (Platform for
Drug Discovery, Informatics, and Structural Life Science)
from the Japan Agency for Medical Research and Develop-
ment (AMED) [to K.I. (1017)].
Conflict of interest
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The authors declare no conflict of interest.
[10] The redox potentials of diselenides (E’0 À381 mV) and seleno-
sulfides (E’0 À326 mV) were measured against DTT (E’0
À323 mV), and that of disulfides (E’0 À215 mV) was measured
against glutathione (E’0 À215 mV) as determined in a model
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Dierks, H. Kessler, L. Moroder, Angew. Chem. Int. Ed. Engl.
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Keywords: amino acids · drug discovery · protein folding ·
selenium · structure elucidation
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Final Article published: && &&, &&&&
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Communications
Protein Folding
Long-lasting insulin, the selenium ana-
logue [selenoinsulin (Se-Ins)] of bovine
pancreatic insulin (BPIns), is
K. Arai, T. Takei, M. Okumura,
S. Watanabe, Y. Amagai, Y. Asahina,
L. Moroder, H. Hojo,* K. Inaba,*
a [C7UA,C7UB] variant and has a structure
and a bioactivity comparable to that of
BPIns. Degradation of Se-Ins with insulin-
degrading enzyme (IDE) was significantly
decelerated (t1/2 ꢀ8 h vs. ꢀ1 h for
M. Iwaoka*
&&&& — &&&&
Preparation of Selenoinsulin as a Long-
Lasting Insulin Analogue
BPIns). The lifetime enhancement is not
only a result of the intrinsic stability of
À
a Se Se bond but also to the local
conformational changes induced by the
substitution.
6
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Angew. Chem. Int. Ed. 2017, 56, 1 – 6
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