Conformationally restricted short peptides inhibit human islet amyloid polypeptide (hIAPP) fibrillization

Article abstract of DOI:10.1039/c3cc38982k

hIAPP fibrillization implicated in Type 2 diabetes pathology involves formation of oligomers toxic to insulin producing pancreatic β-cells. We report design, synthesis, 3D structure and functional characterization of dehydrophenylalanine (ΔF) containing p

Full text of DOI:10.1039/c3cc38982k

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Conformationally restricted short peptides inhibit
human islet amyloid polypeptide (hIAPP) fibrillization†
Cite this: Chem. Commun., 2013,
49, 2688
a
b
Aseem Mishra,z Anurag Misra,z T. Sri Vaishnavi,a Chaitanya Thota,a
Received 16th December 2012,
Accepted 11th February 2013
Madhvi Gupta,a Suryanarayanarao Ramakumar*b and Virander Singh Chauhan*a
DOI: 10.1039/c3cc38982k
www.rsc.org/chemcomm
hIAPP fibrillization implicated in Type 2 diabetes pathology involves to interact with helical monomers of hIAPP. We also propose a
formation of oligomers toxic to insulin producing pancreatic b-cells. We model for fibrillization inhibition by these peptides.
report design, synthesis, 3D structure and functional characterization
Among the core fibrillization motifs/fragments of hIAPP,10
of dehydrophenylalanine (DF) containing peptides which inhibit hIAPP hIAPP(22–27), i.e. NFGAIL, has been shown to form amyloid
fibrillization. The inhibitor protects b-cells from hIAPP induced toxicity. fibrils similar to those formed by the full-length polypeptide.11
Based on the motif hIAPP(22–27), we designed several peptides as
Type 2 Diabetes Mellitus (T2DM) is one of the most prevalent possible inhibitors of hIAPP fibrillization by strategically incorpor-
endocrine disorders underlining the importance of developing mole- ating a non-natural amino acid a,b-dehydrophenylalanine (DF). DF
cular therapies to mitigate T2DM.1 It is characterized by a significant is an analogue of phenylalanine with a double bond between Ca
decrease in b-cell mass, insulin resistance and presence of amyloid and Cb atoms and its presence induces b-turn in short peptides
plaques2 in which human islet amyloid polypeptide (hIAPP) is the and helical secondary structures in longer peptides.12 Also,
major protein component.3 hIAPP is a 37-residue polypeptide peptides containing DF resist enzymatic proteolysis,13 an added
co-secreted with insulin in b-cells of islets of Langerhans. A large advantage for inhibitor design.
amount of evidence favors its wide role in glucose metabolism.4
NFGAIL contains two b-favoring residues, F23 and I26, and their
hIAPP is known to form amyloid fibrils with cross-beta structure,5 replacement with the helicogenic residue DF, individually or
and amyloid deposits as the product of aggregation, but the process together, was a preferred choice for inhibitor design. I26 is an
proceeds through oligomerization.6,7 It has been suggested that important residue; I26 - P mutation in full length hIAPP resulted
hIAPP oligomers of pore-like morphology are formed by association in a hIAPP fibrillization inhibitor.14 Designed peptides (Table S1,
of helical monomers which then perform membrane fragmentation ESI†) were synthesized using solid phase methods, purified on
by pore formation.8 Thus, these prefibrillar oligomers are considered reverse phase HPLC and their identity confirmed by mass spectro-
to be toxic and are implicated in b-cell dysfunction and death.8b,9 scopy (ESI†). Fibrillization was quantified by the enhancement of
Hence, the impairment of oligomerization of helices by using thioflavin T (ThT) fluorescence upon its binding to fibrils. The %
designed small molecule inhibitors such as short peptides is a fibrillization inhibition activities are presented in Table S1 (ESI†).
therapeutically relevant strategy for the prevention of T2DM. In this
I26 - DF mutation in the fibrillizing motif resulted in penta-
report, we show that two pentapeptides related to one of the core and hexapeptides, FGADFL and NFGADFL, respectively. Neither of
fibrillization regions of hIAPP inhibit fibril formation of hIAPP. the two peptides showed b-sheet conformation and fibrillization
Crystal structure analysis revealed an anion receptor ‘nest’ motif in property. ThT assay revealed (Table S1, ESI†) that FGADFL inhi-
these inhibitors, which based on computational studies was shown bited hIAPP fibrillization much more efficiently (75 ꢀ 8%) than
NFGADFL (7 ꢀ 5%). Therefore, we focussed further studies on
a International Centre for Genetic Engineering and Biotechnology,
FGADFL. The fibrillization kinetics of hIAPP in the presence of the
pentapeptide was studied. The exponential increase in ThT inten-
sity, considered as a hallmark of fibril formation, was suppressed
Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail: virander@icgeb.res.in;
Fax: +91-11-26742316; Tel: +91-11-26741358
b Department of Physics, Indian Institute of Science, Bangalore 560012, India.
greatly when hIAPP was incubated with FGADFL in 1 : 5 molar ratio
E-mail: ramak@physics.iisc.ernet.in; Tel: +91-80-22932312
† Electronic supplementary information (ESI) available: Experimental proce- (Fig. 1a) suggesting that the peptide probably curtailed fibrilliza-
dures, list of all the synthesized peptides and their % hIAPP fibrillization
tion at the stage of pre-fibrillar intermediates. Transmission
electron microscopy (TEM) studies also confirmed that FGADFL
significantly decreased hIAPP fibril formation (Fig. 1b and c).
inhibition, MTT cytotoxicity assay, crystallization, details of X-ray structure
determination, in silico docking of FGADFI with hIAPP, CD studies, Tables S1–S4,
and Fig. S1–S6. CCDC 822015 and 904790. For ESI and crystallographic data in
To explore the structure–function relationship, we determined
CIF or other electronic format see DOI: 10.1039/c3cc38982k
‡ These authors contributed equally to this work.
the 3D structure of F1–G2–A3–DF4–L5 through X-ray crystallography
c
2688 Chem. Commun., 2013, 49, 2688--2690
This journal is The Royal Society of Chemistry 2013

View Article Online
Communication
ChemComm
Fig. 3 (a) The best docked pose of hIAPP with FGADFL. (b) Helical wheel
diagram of hIAPP13–30 showing the hydrophobic patch and residues with small
side chains which interact with FGADFL.
Fig. 1 (a) Kinetics of hIAPP fibrillization in the presence and absence of inhibitors.
Time course of amyloid formation monitored by fluorescence detected thioflavin-T
binding: wild-type hIAPP alone (green) and coincubated with 5 M excess of the
designed inhibitors FGADFL (blue) and FGADFI (red). Transmission electron micro-
graphs of (b) hIAPP aged for 40 h, (c) incubated with FGADFL, and (d) FGADFI.
side chain carbonyl/hydroxyl oxygens from hIAPP to satisfy the
hydrogen bond accepting potential of the motif. DF4 in FGADFL was
involved in aromatic p–p stacking interaction with the hIAPP–F23
ring and two clusters of hydrophobic interactions were formed, F1L5
(peptide) & L16V17 (hIAPP) and DF4 (peptide) & F23L27 (hIAPP).
Circular dichroism (CD) spectra of hIAPP in the presence and
absence of inhibitors at two different stages of the hIAPP fibrilli-
zation process i.e. when the hIAPP was in prefibrillar form (3 h old
hIAPP) (Fig. S5, ESI†) and when it reached its fibrillar stage (96 h
old hIAPP) (Fig. S6, ESI†) were recorded and plotted as additive
and complex spectra. CD spectra remained unchanged when
FGADFL was added to hIAPP when it had already attained b-form.
In contrast, the addition of inhibitors at the prefibrillar state of
hIAPP demonstrated noticeable differences in the spectra that
may be because of inhibitor’s binding at the prefibrillar stage.
hIAPP is known to be highly cytotoxic to pancreatic cells.4 In
order to test whether the FGADFL would reduce the cytotoxity of
hIAPP we carried out cell viability assay. Results of the MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay revealed that cytotoxic effects of hIAPP on cultured
pancreatic rat insulinoma cells (RIN 5fm) showed a noticeable
reduction in the presence of FGADFL. The inhibitor on its own
did not show any cytotoxic effects on the cell line.
To further probe structural requirements involved in binding of
the inhibitor with hIAPP, we also synthesized FGADFI, an analogue
of the pentapeptide in which L26 was replaced with a b branched
amino acid, I, and determined its 3D structure through X-ray
crystallography (Fig. S1 and Table S2, ESI†). ThT binding assay
showed that FGADFI was as effective as FGADFL in fibrillization
inhibition (B70% inhibition) (Table S1, ESI†). TEM of hIAPP
incubated with FGADFI showed numerous vesicular structures
without any significant amyloid fibers (Fig. 1d). It was gratifying
to note that although FGADFI crystallized in a different space group
P21 from that of FGADFL (i.e. P212121) (Table S2, ESI†), it also had an
anion receptor nest-motif and superposed with a backbone RMSD
of 0.123 Å with FGADFL (Fig. S2, ESI†). Our in silico studies showed
that both the peptides were able to dock to the monomeric helical
state of hIAPP (ESI†). Perhaps, the interactions involving the anion
receptor together with hydrophobic interactions play a role in the
inhibitory activity of the pentapeptides. However, additional modes
of interactions may also occur since hIAPP is a flexible molecule.
Based on our in silico studies, we propose a possible model
to explain the inhibition of hIAPP fibrillization by the peptides
(Fig. 4). The inhibitors seem to act by binding to the monomeric
(Table S2, ESI†). In the molecule G2 and A3 showed aL and aR
conformations, respectively, and DF4 showed nearly helical con-
formation. Interestingly, a special structural motif ‘nest’ was
observed in the molecule (Fig. 2). In this, two consecutive
N-terminal residues (G2–A3) were positioned such that their main
+
chain NH groups including free NH3 formed a concave depres-
sion serving as an anion receptor, which interacted with terminal
carboxyl oxygen from a symmetry related molecule. The ‘nest’
serves as the binding site of an ‘egg’ which is an atom or a group
of atoms with full or partial negative charge.15 The peptide also
showed a type-I b-turn formed by intramolecular N–Hꢁ ꢁ ꢁO
hydrogen bonding between L5(i+3) - G2(i) (Tables S3 and S4, ESI†).
To investigate the possible modes of interaction of FGADFL with
hIAPP, we performed molecular docking of FGADFL with the 3D
structure of hIAPP (PDB: 2KB8)16 using AutoDock4.17 The best
docked pose resulted in the binding energy of ꢂ6.47 kcal molꢂ1
(Fig. 3a). In the docked complex, FGADFL bound to the stretch
which includes one of the core fibrillization motifs at the C-terminal
half helical region i.e. SNNFGAIL (hIAPP20–27) with a shape com-
plementarity value (Sc)18 of 0.83 indicating that hIAPP and the
inhibitor have complementary binding surfaces. A helical wheel plot
of hIAPP13–30 (Fig. 3b) shows that the face containing small sized
residues (G and S) could easily be approached by the inhibitor.
Docking studies suggested that the nest-motif formed by the FGA
stretch of the pentapeptide interacted with the main chain and/or
Fig. 2 Molecular conformation of FGADFL. (a) Type-I b-turn and (b) anion
receptor nest-motif. Leu side chain observed in two alternative conformations
(a: green, b: yellow). (Details are given in ESI†.)
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 2688--2690 2689

View Article Online
ChemComm
Communication
We thank Dr Patole for cell culture work at NCCS, Pune,
India, and CCD diffractometer facility at IISc. Aseem thanks
Wellcome Trust–DBT India Alliance and Anurag thanks the
University Grants Commission, India, for Research Fellow-
ships. MG thanks Department of Science and Technology
(DST), Govt. of India and ICGEB, VSC thanks Department of
Biotechnology, Govt. of India and SR thanks DST for support.
We thank the reviewers for their valuable comments.
Notes and references
1 P. Hossain, B. Kawar and M. El Nahas, N. Engl. J. Med., 2007, 356, 213.
2 (a) A. E. Butler, J. Janson and S. Bonner-Weir, et al., Diabetes, 2003,
52, 102; (b) A. Clark, C. A. Wells and I. D. Buley, et al., Diabetes Res.,
1988, 9, 151.
3 (a) P. Westermark, C. Wernstedt and E. Wilander, et al., Proc. Natl.
Acad. Sci. U. S. A., 1987, 84, 3881; (b) G. J. S. Cooper, A. C. Willis and
A. Clark, Proc. Natl. Acad. Sci. U. S. A., 1987, 84, 8628.
4 P. Westermark, A. Andersson and G. T. Westermark, Physiol. Rev.,
2011, 91, 795.
5 J. J. Wiltzius, S. A. Sievers and D. Eisenberg, et al., Protein Sci., 2008,
17, 1467.
6 M. Magzoub and A. D. Miranker, FASEB J., 2012, 26, 1228.
7 A. Laganowsky, C. Liu and M. R. Sawaya, et al., Science, 2012, 335, 1228.
8 (a) R. Soong, J. R. Brender and P. M. Macdonald, et al., J. Am. Chem. Soc.,
2009, 131, 7079; (b) J. R. Brender, S. Salamekh and A. Ramamoorthy,
Acc. Chem. Res., 2012, 45, 454.
Fig. 4 A proposed model for the inhibition of hIAPP fibrillization by FGADFL/
FGADFI. (a) Unfolded state of hIAPP; (b) monomeric helical form of hIAPP (2KB8);
(c) 3D structure of the inhibitor FGADFL, stick and surface representations;
(d) inhibitor computationally docked with hIAPP; complex structure. The binding
of inhibitors to hIAPP is expected to discourage the formation of toxic oligomers/
fibrils (e), which in the final stage of aggregation result in amyloid plaques (f).
helical state of hIAPP at the hIAPP20–27 region which is one of
the core fibrillization regions of hIAPP implicated in the
9 L. Haataja, T. Gurlo and C. J. Huang, et al., Endocr. Rev., 2008, 29, 303.
nucleation dependent mechanism for oligomerization and 10 X. B. Mao, C. X. Wang and X. K. Wu, et al., Proc. Natl. Acad. Sci. U. S. A.,
2011, 108, 19605.
initiation of beta-sheet formation during the fibrillization
11 K. Tenidis, M. Waldner and J. Bernhagen, et al., J. Mol. Biol., 2000,
event.19 Binding of the inhibitor would stabilize the monomeric
295, 1055.
helical form of hIAPP and would decrease contact between 12 (a) K. R. Rajashankar, S. Ramakumar and V. S. Chauhan, J. Am.
Chem. Soc., 1992, 114, 9225; (b) K. R. Rajashankar, S. Ramakumar
and T. K. Mal, et al., Angew. Chem., Int. Ed. Engl., 1996, 35, 765;
(c) P. Mathur, S. Ramakumar and V. S. Chauhan, Biopolymers, 2004,
helices coming in the way of helix–helix association. This would
reduce the possibility of oligomer formation and fibrillization.
hIAPP fibrillization inhibitors include organic molecules,20 frag-
ments of hIAPP21,22 as well as variants of native protein14,23–25 and
its fragments.26–28 An approach in these studies was to disrupt
amyloid formation by hIAPP and include peptides containing beta-
breaker residues like Aib, Pro and N-methylated residues.14,23,28 In
many cases, attention has been focused on targeting fibril for-
mation by reducing b-sheet extension and assembly. Targeting
amyloid fibrils may not be a useful strategy due to an adverse
effect of rapidly increasing oligomers.29 Since FGADFL binds to
hIAPP in the monomeric state, it could, in principle, shift the
equilibrium away from the formation of oligomers/intermediates
toxic to beta cells. Therefore, targeting the transient monomeric
helical state and discouraging helix–helix association leading to the
formation of oligomeric nuclei in the early events of self assembly,
as outlined here, may be an attractive strategy for inhibitor design.
To conclude, though it was not anticipated, crystal structure
analysis revealed that FGADFL and its analogue FGADFI harbour
the anion receptor ‘nest’ motif. Both peptides dock with the
helical form of hIAPP which may contribute to the inhibitory
function of the peptides through their interaction with hIAPP in
the core fibrillization region. These peptides effectively inhibit
hIAPP fibrillization in vitro and it seems that these are unique
examples of nest-motif containing peptides that inhibit fibrilli-
zation. In general, the approach described here may be applic-
able to targeting helices or helical intermediates and could be
utilized in developing inhibitors useful, apart from T2DM, in
other amyloid diseases including Alzheimer’s disease and
Parkinson’s disease.30
76, 150; (d) U. A. Ramagopal, S. Ramakumar and D. Sahal, et al.,
Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 870; (e) M. Gupta, R. Acharya
and A. Mishra, et al., ChemBioChem, 2008, 9, 1375.
13 M. L. English and C. H. Stammer, Biochem. Biophys. Res. Commun.,
1978, 83, 1464.
14 A. Abedini, F. Meng and D. P. Raleigh, J. Am. Chem. Soc., 2007,
129, 11300.
15 (a) J. D. Watson and E. J. Milner-White, J. Mol. Biol., 2002, 315, 171;
(b) Rudresh, S. Ramakumar and U. A. Ramagopal, et al., Structure,
2004, 12, 389.
16 S. M. Patil, S. Xu and S. R. Sheftic, et al., J. Biol. Chem., 2009, 284, 11982.
17 (a) G. M. Morris, D. S. Goodsell and R. S. Halliday, et al., J. Comput.
Chem., 1998, 19, 1639; (b) G. M. Morris, R. Huey and W. Lindstrom,
et al., J. Comput. Chem., 2009, 30, 2785.
18 M. C. Lawrence and P. M. Colman, J. Mol. Biol., 1993, 234, 946.
19 J. J. W. Wiltzius, S. A. Sievers and M. R. Sawaya, et al., Protein Sci.,
2009, 18, 1521.
20 J. A. Hebda, I. Saraogi and M. Magzoub, et al., Chem. Biol., 2009,
16, 943.
21 L. A. Scrocchi, Y. Chen and S. Waschuk, et al., J. Mol. Biol., 2002,
318, 697.
22 K. J. Potter, L. A. Scrocchi and G. L. Warnock, et al., Biochim.
Biophys. Acta, 2009, 1790, 566.
23 L. M. Yan, M. Tatarek-Nossol and A. Velkova, et al., Proc. Natl. Acad.
Sci. U. S. A., 2006, 103, 2046.
24 F. Meng, D. P. Raleigh and A. Abedini, J. Am. Chem. Soc., 2010,
132, 14340.
25 J. R. Cort, Z. Liu and G. M. Lee, et al., Protein. Eng., Des. Sel., 2009,
22, 497.
26 A. Kapurniotu, A. Schmauder and K. Tenidis, J. Mol. Biol., 2002,
315, 339.
27 M. Tatarek-Nossol, L. M. Yan and A. Schmauder, et al., Chem. Biol.,
2005, 12, 797.
28 S. Gilead and E. Gazit, Angew. Chem., Int. Ed., 2004, 43, 4041.
29 J. A. Hebda and A. D. Miranker, Annu. Rev. Biophys., 2009, 38, 125.
30 J. D. Sipe, Amyloid Proteins: The b-Sheet Conformation and Disease,
Wiley-VCH, Weinheim, 2005.
c
2690 Chem. Commun., 2013, 49, 2688--2690
This journal is The Royal Society of Chemistry 2013