Rational design of an orthosteric regulator of hIAPP aggregation

Article abstract of DOI:10.1039/c4cc06739h

Developing compounds regulating amyloid toxic oligomer but not fibril formation should constitute an effective strategy for the treatment of diabetes. Based on the full understanding of the folding mechanism, we designed an orthosteric helix regulator that can promote hIAPP to assemble into large non-cytotoxic oligomers. As a result, the islet cells were protected. This journal is

Full text of DOI:10.1039/c4cc06739h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Rational design of an orthosteric regulator of
hIAPP aggregation†
De-Sheng Zhao,a Yong-Xiang Chena and Yan-Mei Li*ab
Cite this: Chem. Commun., 2015,
51, 2095
Received 27th August 2014,
Accepted 6th December 2014
DOI: 10.1039/c4cc06739h
www.rsc.org/chemcomm
Developing compounds regulating amyloid toxic oligomer but not fibril
Numerous investigations have suggested that amyloid oligomers
formation should constitute an effective strategy for the treatment are b-sheet-rich structures.20,21 Additionally, a b-strand-rich dimer
of diabetes. Based on the full understanding of the folding mechanism, has been included in the aggregation pathway as this structure
we designed an orthosteric helix regulator that can promote hIAPP appears to have the greatest likelihood of forming oligomers.22,23
to assemble into large non-cytotoxic oligomers. As a result, the islet Our converged replica exchange molecule dynamic (REMD) simula-
cells were protected.
tions suggested that the b-strand-rich dimers were formed through
an a-helix-to-b-sheet transition mechanism (Fig. 1a). The hIAPP
The human islet amyloid polypeptide (hIAPP), also known as monomer may adopt a variety of conformations and it is very
amylin, is a 37-residue peptide. It is produced by the b-cells along difficult for the hIAPP monomer to misfold into b-rich conforma-
with insulin and plays an important role in controlling gastric tions directly, as the potential energy barrier is too high and the
emptying, glucose homeostasis, and suppression of glucagon energy basins are very shallow (Fig. S4 in ESI†). Because the helix
release.1 In 1987, hIAPP was found to be the primary component intermediates are the prerequisite and rate determining step in the
of the amyloid deposits around the b-cells in patients with Type 2 formation of b-strand-rich dimers, which once formed, will rapidly
diabetes. The formation of the islet amyloid can lead to b-cell and easily assemble into oligomers, we reasoned that artificial
dysfunction, death, and reduction in b-cell mass.2 To prevent the a-helices that mimic hIAPP could enhance the formation of helix
b-cell from being harmed, inhibitors have been designed recently intermediates and markedly promote hIAPP aggregation. By
and found to markedly prevent the formation of islet amyloid chaperoning IAPP through fusion to the maltose binding protein,
fibrils.3–10 However, a growing body of evidence has identified the crystal structure of the hIAPP helix dimer has been obtained.24
amyloid oligomers formed in the early stage as the basic cytotoxic Computational analysis identified Leu12 and Phe15 as residues
components of the aggregation pathway11–15 and most of these that contribute most strongly to the interaction of the hIAPP helix
inhibitors have little effect on the formation of hIAPP toxic dimer (Fig. 1b). Thus, we designed a stable helix peptide which
oligomers. Besides, oligopyridylamide a-helix mimetics have been mimics the fragment hIAPP12–15. To stabilize the short peptide in
designed as agonists and antagonists of hIAPP fibril formation.16 a-helical conformation, the hydrogen bond surrogate (HBS)
However, whether the oligopyridylamide compound can regulate approach has been adopted (Fig. 1d) in which the hydrogen bond
hIAPP oligomer formation remains unknown. Encouragingly, for between the carboxyl of ith residues and the amide of i + 4th
b-amyloid and insulin, new inhibitors that target the amyloid residues is surrogated by carbon–carbon bonds. We chose HBS
folding intermediate in the early stage have been successfully helices as they have shown high affinity and specificity previously
rationally designed.17–19 Accordingly, the design of new inhibitors when targeted to protein receptors.25,26
targeting the intermediates in the early stage and blocking the
We optimized the sequence using a sequence tolerance
formation of toxic hIAPP oligomers should constitute an effective method.27 L12 and F15 were proven to be the best choices to
strategy for the treatment of Type 2 diabetes.
enhance the helix–helix interaction. It corresponds to the native
peptide. We proposed to replace L16 with glutamic acid to enhance
the solubility and to stabilize the a-helical conformations (Fig. 1c).
Further CD spectroscopy studies on control peptides (mutation of
hot spot residues to alanine) showed that these residues are
essential to stabilize the helical conformation (Fig. S7 in ESI†).
Alanine was used as the backbone for its high helix-forming
propensity. To further increase the contents of helix structures,
a Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry &
Chemical Biology (Ministry of Education), Tsinghua University, Beijing 10084,
P. R. China. E-mail: liym@mail.tsinghua.edu.cn; Fax: +86-10-62781695
b Beijing Institute for Brain Disorders Center of Parkinson’s Disease, P. R. China
† Electronic supplementary information (ESI) available: Supplementary materi-
als, methods, results, and references. See DOI: 10.1039/c4cc06739h
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 2095--2098 | 2095

View Article Online
Communication
ChemComm
helical in PBS only (Fig. S7 in ESI†). The high docking score and
low docking RMSD values when HBS 1 was docked to hIAPP
suggested that HBS 1 can bind to hIAPP efficiently (Fig. S6 in
ESI†). The free energy surface of HBS 1 suggested that it could
fold to the helix conformation with high probability (Fig. S8 in
ESI†). When assayed in a Thioflavin T (ThT) fluorescence
kinetic study, the designed compound promoted hIAPP aggre-
gation (Fig. 2a). In the presence of HBS 1, hIAPP quickly
assembled to an intermediate with a high ThT fluorescence
signal. These intermediates then transformed slowly to another
aggregation state with a decrease of ThT fluorescence intensity.
Furthermore, 10% TFE was used as a positive control to induce the
amyloid aggregate through an a-helix aggregation mechanism.31
We also found that in the presence of 10% TFE, hIAPP can
assemble rapidly to an intermediate with high ThT fluorescence
intensity and could then transfer to other aggregation states
and attain a plateau (Fig. 2a). Circular dichroism spectroscopy
suggested that addition of HBS 1 or trifluoroethanol can
promote hIAPP folding to helix-rich intermediates firstly (Fig. 2b),
followed by conversion to b-rich structures (Fig. 2c). Besides, during
the process of hIAPP aggregation, HBS 1 maintained its helix
conformation and could not interact with ThT (Fig. S12 and S14c
in ESI†). For more details about conformation transition analysis,
see ESI.† We determined that enhancing the helix intermediates
may promote the formation of hIAPP aggregates with high ThT
fluorescence intensity. However, there were no intermediates formed
when hIAPP was prepared with the analog of HBS 1, UC 1, whose
sequence was the same as HBS 1, but lacking a constrained
conformation (Fig. S11 in ESI†).
To clarify the cause of the high ThT fluorescence intensity
when HBS 1 was added to the system, we performed morpho-
logic studies on these aggregation intermediates. We found that
in the presence of HBS 1, hIAPP tended to assemble into large
globular oligomers (Fig. 2g). The diameter was about 600 nm
which corresponded to the Dynamic Light Scattering measure-
ment (DH = 700 nm) (Fig. 2h). However, in the absence of HBS 1,
hIAPP formed small oligomers with a diameter of about 80 nm
(Fig. 2e). This value also corresponded to the Dynamic Light
Scattering measurement (DH = 90 nm) (Fig. 2f). Both of these
peptides will eventually convert to fibers (Fig. S13 in ESI†). We
propose that the increase of ThT fluorescence intensity is caused
by the large globular oligomers formed in the presence of HBS 1.
The transition from these large globular aggregates to fibrils
leads to the decrease of ThT fluorescence intensity and finally
results in the same kinetic curve as hIAPP only.
Fig. 1 The helix–helix interaction of the hIAPP dimer and rational design of
the synthetic peptide. (a) Two-dimensional free energy maps of the hIAPP7-29
dimer at 300 K as a function of average Psi angle of only one chain and the
root mean square deviation (nm). Representative structures in each minimum-
energy basin are also given: (I) (0.444, À41.75); (II) (0.46, À25.25); (III) (0.7425,
56.45); (IV) (0.80, 63.25); (V) (0.98, 109.15); the red line represents the
conformation transition pathway. (b) Crystal structure of the maltose binding
protein (MBP) – hIAPP fusion proteins (PDB code: 3G7V). Virtual alanine
scanning suggests that hot spots of the helix dimer are L12 and F15 (inset).
(c) The hIAPP helix–helix interface tolerance prediction. The optimized
sequence was XLXXFEX (X represents any amino acid). (d) The hydrogen bond
surrogate (HBS) helices structure. The intra-molecular hydrogen bond
between i and i + 4 residues has been replaced with a covalent carbon–
carbon bond. The sequence of the optimized hIAPP mimetic HBS 1 is shown.
X represents a 4-pentenoic acid residue. Me represents methyl.
To explain how these large oligomers formed, we investigated
the formation of oligomers using enlargements of TEM images,
which indicated that these large oligomers were formed by further
assembly of some aggregated units (Fig. S17 in ESI†). By perform-
the C-terminus was modified with N-methylamine. This resulted ing photo-induced cross-linking of unmodified proteins (PICUP)
in the optimized sequence: ALAAFEA-NH-Me. For more informa- and sodium dodecyl sulfate polyacrylamide gel electrophoresis
tion of the a-helix-to-b-sheet transition mechanism and peptide (SDS-PAGE) with silver staining (Fig. 2d), we found that in the
design strategy, see ESI.† The HBS helices were synthesized as presence of HBS 1, hIAPP formed large aggregated units (molecule
previously reported28–30 (Fig. S1 and S2 in ESI†).
weight is B70 kDa;) quickly. This result corresponded to a
The optimized HBS helix, HBS 1 (Fig. 1d), was judged to be typical helix aggregation mechanism in which helix monomers
83% helical in 10% trifluoroethanol (TFE) in PBS and 67% will assemble into large aggregate units easily and enhance the
2096 | Chem. Commun., 2015, 51, 2095--2098
This journal is ©The Royal Society of Chemistry 2015

View Article Online
ChemComm
Communication
Fig. 3 Effect of HBS 1 on the interaction of hIAPP with the membrane.
(a) Dye leakage assay from 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine
(POPC) and 2-oleoyl-1-palmitoyl-sn-glycero-3-glycerol (POPG) (3 : 1) vesicles
(15.68 mM) induced by full length hIAPP (hIAPP, 1 mM; blue line), hIAPP (1 mM) +
HBS1 (10 mM) (green line), HBS 1 (10 mM; red line). (b) Dye leakage assay induced
by hIAPP1-19 (1 mM; blue line) and hIAPP1-19 (1 mM) + HBS 1 (10 mM) (red line).
(c) The viability of INS-1 rat insulinoma cells treated with hIAPP (1 mM, 5 mM,
10 mM) or hIAPP (1 mM, 5 mM, 10 mM) + HBS1 (10 mM). The level of significance
indicated by *** p o 0.001; ** p o 0.01; and * p o 0.05 (by 2-tailed Student’s
t-test). The data represent the mean Æ S.D. (n = 4).
units quickly (Fig. S15 and S16 in ESI†). Its morphology corre-
sponded to the TEM studies. That is to say, HBS 1 promoted the
formation of hIAPP large aggregate units which further assembled
into large globular oligomers. It is these large globular oligomers
that caused the high fluorescence intensity observed in the
ThT assay.
To identify whether promoting the formation of large globular
oligomers will prevent cytotoxicity induced by the small hIAPP
oligomers, we explored the effect of HBS 1 on the interaction
between hIAPP and the membrane. We found that HBS 1 decreased
the relative dye leakage percentage induced by hIAPP by 40% (20%
for ratio 1 : 1; 50% for ratio 1 : 100; Fig. S20 in ESI†) (Fig. 3a) at
20 min and by 60% for leakage induced by hIAPP1-19 at 300 s (Fig. 3b),
indicating that promoting the formation of large oligomers can
indeed inhibit hIAPP toxicity. On the other hand, the unconstrained
control peptide UC 1 slightly decreased the relative dye leakage
percentage (Fig. S21 in ESI†). Besides, from the comparison of the
extended dye leakage experiment and ThT experiment, we believed
that HBS 1 mainly functioned by blocking oligomer mediated
membrane damage (Fig. S18 and S19 in ESI†).
Fig. 2 Effect of HBS 1 on hIAPP aggregation kinetic behaviors. (a) ThT
fluorescence of hIAPP only (blue line, 10 mM), in the presence of HBS 1 (red
line, 100 mM) and in the presence of 10% TFE (green line). (b) CD spectrum
of hIAPP helix intermediates. Data were measured immediately after
peptide dissolution. Blue line: hIAPP (10 mM); red line: hIAPP (10 mM) +
HBS 1 (100 mM) absorbance induced by HBS 1 was subtracted; green line:
hIAPP (10 mM) in 10% TFE. (c) CD spectrum of hIAPP b-sheet conformation
after incubation for 140 min. Blue line: hIAPP (10 mM); red line: hIAPP
(10 mM) + HBS 1 (100 mM) absorbance induced by HBS 1 was subtracted;
green line: hIAPP (10 mM) in 10% TFE. (d) The peptide composition of hIAPP
(30 mM) in the absence and presence of HBS 1 (300 mM) monitored by PICUP
SDS-PAGE-silver staining. For lane (A): marker; (B) hIAPP 30 mM, 20 min;
(C): hIAPP 30 mM, 80 min; (D): hIAPP 30 mM, 140 min; (E): hIAPP 30 mM,
200 min; (F): hIAPP 30 mM and HBS 1 300 mM, 20 min; (G): hIAPP 30 mM and
HBS 1 300 mM, 80 min; (H): hIAPP 30 mM and HBS 1 300 mM, 140 min;
(I): hIAPP 30 mM and HBS 1 300 mM, 200 min; (e) the TEM image of hIAPP
small oligomers after incubation for 60 min. (hIAPP: 10 mM) (f) DLS analysis
of small oligomers formed by hIAPP only after incubation for 60 min (hIAPP:
10 mM). (g) The TEM image of large globular oligomers in the presence of
HBS 1 after incubation for 60 min. (hIAPP: 10 mM; HBS 1: 100 mM). (h) DLS
analysis of large globular oligomers formed by hIAPP in the presence of HBS
1 after incubation for 60 min. (hIAPP: 10 mM; HBS 1: 100 mM).
Cell viability assays established that HBS 1 can indeed reduce
the toxicity of hIAPP on INS-1 cells (Fig. 3c). These assays showed
that the hIAPP pre-incubated without HBS 1 killed about 70% of
transition to b-rich aggregates.32 However, for hIAPP, we only the INS-1 cells, relative to controls, when cells were incubated
observed some small aggregate units (monomers, dimers and a in PBS buffer solutions only. However, cell survival increased
few trimers). Coarse-grain MD simulations also suggested that notably when cells were cultured with a pre-incubated mixture of
in the absence of HBS 1, hIAPP tends to aggregate to small HBS 1 and hIAPP at different concentration. Furthermore,
aggregate units (dimer and trimer). However, in the presence of we found that HBS 1 prevented hIAPP from changing the cell
HBS 1, hIAPP will co-assemble with HBS 1 to large aggregate morphology from a native fusiform state to a transparent
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun., 2015, 51, 2095--2098 | 2097

View Article Online
Communication
ChemComm
8 A. Mishra, A. Misra, T. S. Vaishnavi, C. Thota, M. Gupta, S. Ramakumar
and V. S. Chauhan, Chem. Commun., 2013, 49, 2688–2690.
9 L. M. Yan, M. Tatarek-Nossol, A. Velkova, A. Kazantzis and
A. Kapurniotu, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 2046–2051.
10 J. Hu, Y. P. Yu, W. Cui, C. L. Fang, W. H. Wu, Y. F. Zhao and Y. M. Li,
Chem. Commun., 2010, 46, 8023–8025.
11 G. J. S. Cooper, J. F. Aitken and S. Zhang, Diabetologia, 2010, 53,
1011–1016.
12 J. R. Brender, S. Salamekh and A. Ramamoorthy, Acc. Chem. Res.,
2012, 45, 454–462.
13 R. A. Ritzel, J. J. Meier, C. Y. Lin, J. D. Veldhuis and P. C. Butler,
Diabetes, 2007, 56, 65–71.
14 Y. P. Yu, P. Lei, J. Hu, W. H. Wu, Y. F. Zhao and Y. M. Li, Chem.
Commun., 2010, 46, 6909–6911.
15 M. S. Chen, D. S. Zhao, Y. P. Yu, W. W. Li, Y. X. Chen, Y. F. Zhao and
Y. M. Li, Chem. Commun., 2013, 49, 1799–1801.
ball state. Perhaps HBS 1 blocked organelle leakage from a cell
(Fig. S22 in ESI†).
Using our understanding of the a-helix-to-b-sheet transition
mechanism as a basis for our design, we synthesized a helix
mimetic regulator which promoted the formation of large nontoxic
oligomers and reduced the cytotoxicity induced by small oligomers.
We suggest the targeting of the helix intermediates in the very early
stage of the aggregation as an alternative therapeutic approach to
amyloid diseases. This study not only provided a strategy for
inhibitor design based on the helix intermediates of hIAPP but
also revealed some clues toward understanding the molecular
events involved in hIAPP aggregation.
16 I. Saraogi, J. A. Hebda, J. Becerril, L. A. Estroff, A. D. Miranker and
A. D. Hamilton, Angew. Chem., Int. Ed., 2010, 49, 736–739.
17 Y. Xu, J. Shen, X. Luo, W. Zhu, K. Chen, J. Ma and H. Jiang, Proc.
Natl. Acad. Sci. U. S. A., 2005, 102, 5403–5407.
18 D. Liu, Y. Xu, Y. Feng, H. Liu, X. Shen, K. Chen, J. Ma and H. Jiang,
Biochemistry, 2006, 45, 10963–10972.
19 Y. Hong, L. Meng, S. Chen, C. W. Leung, L. T. Da, M. Faisal,
D. A. Silva, J. Liu, J. W. Lam, X. Huang and B. Z. Tang, J. Am.
Chem. Soc., 2012, 134, 1680–1689.
20 A. Laganowsky, C. Liu, M. R. Sawaya, J. P. Whitelegge, J. Park, M. Zhao,
A. Pensalfini, A. B. Soriaga, M. Landau, P. K. Teng, D. Cascio, C. Glabe
and D. Eisenberg, Science, 2012, 335, 1228–1231.
This work was supported by the major State Basic Research
Development Program of China (973 program) (No. 2013CB910700,
2012CB821600) and grants from the National Natural Science Foun-
dation of China (No. 91313301, 21472109, 21261130090). This work
was supported by Tsinghua National Laboratory for Information
Science and Technology and Beijing Nuclear Magnetic Resonance
Center. We thank Prof. Zhixiang Yu in Peking University for help with
the microwave synthesis, Hong Liu, Dongxiang Liu, Yechun Xu in
Shanghai Institute of Materia Medica Chinese Academy of Sciences
and Dong Wang in Tsinghua University for helpful comments on
molecular dynamics simulation.
21 J. C. Stroud, C. Liu, P. K. Teng and D. Eisenberg, Proc. Natl. Acad. Sci.
U. S. A., 2012, 109, 7717–7722.
22 N. F. Dupuis, C. Wu, J.-E. Shea and M. T. Bowers, J. Am. Chem. Soc.,
2011, 133, 7240–7243.
23 N. F. Dupuis, C. Wu, J. E. Shea and M. T. Bowers, J. Am. Chem. Soc.,
2009, 131, 18283–18292.
24 J. J. Wiltzius, S. A. Sievers, M. R. Sawaya and D. Eisenberg, Protein
Sci., 2009, 18, 1521–1530.
Notes and references
1 G. J. Cooper, Endocr. Rev., 1994, 15, 163–201.
2 P. Westermark, C. Wernstedt, E. Wilander, D. W. Hayden,
T. D. O’Brien and K. H. Johnson, Proc. Natl. Acad. Sci. U. S. A., 25 L. K. Henchey, J. R. Porter, I. Ghosh and P. S. Arora, ChemBioChem,
1987, 84, 3881–3885. 2010, 11, 2104–2107.
3 A. Kapurniotu, A. Schmauder and K. Tenidis, J. Mol. Biol., 2002, 315, 26 L. K. Henchey, S. Kushal, R. Dubey, R. N. Chapman, B. Z. Olenyuk
339–350. and P. S. Arora, J. Am. Chem. Soc., 2010, 132, 941–943.
4 S. Gilead and E. Gazit, Angew. Chem., Int. Ed., 2004, 43, 4041–4044. 27 E. L. Humphris and T. Kortemme, Structure, 2008, 16, 1777–1788.
5 R. Mishra, B. Bulic, D. Sellin, S. Jha, H. Waldmann and R. Winter, 28 R. N. Chapman and P. S. Arora, Org. Lett., 2006, 8, 5825–5828.
Angew. Chem., Int. Ed., 2008, 47, 4679–4682.
6 R. Mishra, D. Sellin, D. Radovan, A. Gohlke and R. Winter,
ChemBioChem, 2009, 10, 445–449.
29 R. N. Chapman, G. Dimartino and P. S. Arora, J. Am. Chem. Soc.,
2004, 126, 12252–12253.
30 D. Wang, K. Chen, J. L. Kulp Iii and P. S. Arora, J. Am. Chem. Soc.,
2006, 128, 9248–9256.
7 J. Zheng, C. Liu, M. R. Sawaya, B. Vadla, S. Khan, R. J. Woods,
D. Eisenberg, W. J. Goux and J. S. Nowick, J. Am. Chem. Soc., 2011, 31 Y. Fezoui and D. B. Teplow, J. Biol. Chem., 2002, 277, 36948–36954.
133, 3144–3157.
32 A. Abedini and D. P. Raleigh, Phys. Biol., 2009, 6, 015005.
2098 | Chem. Commun., 2015, 51, 2095--2098
This journal is ©The Royal Society of Chemistry 2015