Synthetic α-helix mimetics as agonists and antagonists of islet amyloid polypeptide aggregation

Article abstract of DOI:10.1002/anie.200901694

Figure Presented Split personality: A series of oligoamidebased helix mimetics bind to a complementary helical motif in Islet amyloid polypeptide (IAPP), a protein implicated in the pathology of type II diabetes. These compounds accelerated IAPP amyloid formation under lipid-free conditions, but inhibited it under lipid-catalyzed conditions. hlAPP = human IAPP.

Full text of DOI:10.1002/anie.200901694

Communications
DOI: 10.1002/anie.200901694
Proteomimetics
Synthetic a-Helix Mimetics as Agonists and Antagonists of Islet
Amyloid Polypeptide Aggregation**
Ishu Saraogi, James A. Hebda, Jorge Becerril, Lara A. Estroff, Andrew D. Miranker,* and
Andrew D. Hamilton*
The development of small molecules that can modulate the
damaging effects of protein aggregation processes remains a
high priority goal in contemporary medicinal chemistry.[1] An
important class of these aggregates, called amyloids, has been
implicated in numerous degenerative diseases including
Alzheimerꢀs, type II diabetes, senile systemic amyloidosis
(SSA), prion diseases, and rheumatoid arthritis. The attribute
shared by these symptomatically unrelated diseases is that a
normally soluble protein undergoes a conformational change
resulting in self-assembly into cytotoxic forms, culminating in
a b-sheet-rich fibrillar structure. Islet amyloid polypeptide
(IAPP), or amylin, is one such protein which has been
implicated in amyloidogenesis in type II diabetes.[2] IAPP is
cosecreted with insulin by the b cells of the islets of
Langerhans and an aggregated form of IAPP is believed to
play a role in b-cell toxicity in the pathology of type II
diabetes.[3]
Amyloid-forming processes proceed by a nucleation
dependent reaction mechanism. The structural and energetic
basis for nucleation is, however, poorly understood.[5] For
IAPP, Knight and Miranker[6] proposed a possible mechanism
where nucleation is initiated by the binding of IAPP to cell
membranes through contacts mediated by residues 1–20
(Figure 1a).[4] The region of IAPP comprising residues 5–19
clearly shows a helical structure having positive charges
predominant on one face, and likely forms multihelical
aggregates upon interaction with the membrane surface.[7,8]
The formation of these a-helical intermediates accelerates the
assembly of the amyloid structure which is rich in b sheets.[9]
Figure 1. a) Model for IAPP amyloid formation with a-helical inter-
mediate states.[4] b) Schematic representation of an a-helix mimetic of
varying length, interacting with the a-helical intermediate in the IAPP
fibril formation pathway.
Recent findings have additionally suggested that these helical
oligomeric intermediates may be the relevant cytotoxic form
of IAPP.[8,10] An interesting, and previously unexplored,
potential therapeutic approach for mitigating the cytotoxic
effects of IAPP would be to design molecules that interfere
with the helix assembly process (Figure 1b).
Inhibition of IAPP aggregation by small molecules based
on a rhodanine scaffold,[11] phenol red,[12] and phenolsul-
fonphthalein[13] has been reported. Similar disruption of
amyloid assembly in other protein aggregation diseases by
aromatic dyes is known.[14,15] A plausible general mechanism
for such inhibition involves p stacking of the dye with the
aromatic amino-acid-rich core of the developing amyloid.[12,15]
In this work, we propose an alternative mechanism of amyloid
inhibition wherein we target the transient a-helical inter-
mediates in IAPP aggregation.
We have previously reported synthetic structures that
mimic the residues along one face of an a helix and
successfully disrupt important protein–protein interactions.[16]
In particular, the oligopyridylamide scaffold 1 uses intra-
molecular hydrogen bonding to rigidify the backbone, and
projects functionality on one face of the molecule in direct
analogy to an a helix.[17–19]
An inspection of the N-terminal region of human IAPP
(hIAPP) reveals four positive charges in close spatial
proximity: Arg11 and His18 (which is likely protonated at
the membrane surface) in the helical domain, as well as Lys1
and the N-terminus. A potential size and charge complemen-
tarity with this region might be achieved by a tetrameric or
pentameric form of the oligopyridylamide scaffold containing
four or five carboxy-terminated side chains, respectively. To
study systematically the effect of an increasing number of
negative charges on interaction with IAPP, the monomeric
[*] J. A. Hebda,[+] Prof. A. D. Miranker
Molecular Biophysics and Biochemistry, Yale University
266 Whitney Avenue, P.O. Box 208114
New Haven, CT 06520-8114 (USA)
Fax: (+1)203-432-3104
E-mail: andrew.miranker@yale.edu
I. Saraogi,[+] J. Becerril, L. A. Estroff, Prof. A. D. Hamilton
Department of Chemistry, Yale University
225 Prospect Street, P.O. Box 208107
New Haven, CT 06520-8107 (USA)
Fax: (+1)203-432-6144
E-mail: andrew.hamilton@yale.edu
[+] These authors contributed equally to the work.
[**] We thank Prof. G. W. Brudvig for suggestions and Dr. C. Incarvito for
assistance with X-ray crystallographic analysis. This work was
supported by National Institutes of Health grants to A.D.H.
(GM69850) and A.D.M. (NIDDK DK079829). J.A.H. was supported
by a NRSA fellowship (AG031612).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200901694.
736
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 736 –739

Angewandte
Chemie
through pentameric pyridylamides 1a–1e were synthesized.
Furthermore, to probe the efficacy of the hydrogen-bonding
preorganization effect in these molecules, the corresponding
oligobenzamide series 2a–2e was synthesized,[18,20,21] in which
the pyridine rings were replaced by benzene so that bifurcated
hydrogen bonding was no longer possible. These molecules
=
have greater conformational flexibility about the aryl-C( O)
bonds and permit an adaptability of structure on binding,
albeit at an entropic cost.
The molecules were synthesized using linear solution-
phase iterative coupling as reported earlier for the shorter
homologues.[18,19] Briefly, chain elongation was achieved using
successive amide coupling and nitro group reduction steps
(see the Supporting Information). The acid groups, which
were protected as tert-butyl esters, were cleaved in the last
step to give the target compounds.
Figure 2. a) X-ray crystal structure of the tert-butylester of 1d (tBu
ester groups and non-NH hydrogen atoms have been omitted for
clarity). b) Crystal structure of the backbone of 1e (Y=NH2), the side
chains could not be modeled because of positional disorder, and are
shown as red balls. Red O, blue N, green C, white H.
The dimers (1b, 2b) and trimers (1c, 2c) have previously
been shown to adopt an elongated rod-like conformation in
the solid state with a more curved backbone in the oligopyr-
idylamides than the oligobenzamides.[18] This effect continues
in tetrameric 1d and pentameric 1e, as seen from their crystal
structures (Figure 2), and the side chains project from one
face of the molecule, forming a recognition surface for
potential binding to a complementary face of an a helix. As a
result of some positional disorder, only four of the five side
chains in the crystal structure of 1e could be modeled.
However, the location of the oxygen atom of the side chains,
as identified from the electron density map, allows us to
conclude that 1e, like its shorter homologues, adopts an
extended curved conformation with the side chains projected
on one face. Molecular modeling studies with 2d and 2e
suggest that they adopt a similar extended conformation, with
less certainty over the position of the side chains.[20]
IAPP binds lipid membranes and its fibrillization is
strongly accelerated in the presence of liposomes containing
mixtures of anionic dioleoylphosphatidylglycerol (DOPG)
and zwitterionic dioleoylphosphatidylcholine (DOPC).[6] The
kinetic profile is characterized by a lag phase with a
subsequent cooperative transition into the aggregated state.
The rate of fibrillogenesis of IAPP under lipid-catalyzed
conditions, in the presence of the helix mimetics, was
determined using an exogenous fluorescent dye, thioflavin-T
(ThT). ThT binds specifically to amyloid fibrils without
significantly disturbing the IAPP fiber formation pathway,[6]
and the relative fluorescence intensity is an indicator of the
transition into the amyloid state. Since compounds that
interact with IAPP and affect fiber formation can interfere
with ThT binding, our analysis of changes in amyloid
formation kinetics was limited to the midpoint of the
transition, t50, rather than the absolute ThT signal.
In the presence of the helix mimetics, the rate of
acceleration of lipid-catalyzed IAPP fiber formation was
significantly diminished. A representative kinetic data plot
for 1e is shown in Figure 3a. Molecule 1e was an effective
inhibitor of the lipid-catalyzed IAPP fiber formation with a
relative t50 that is four-fold higher than the control reaction.
This inhibition was dose-dependent and an IC50 of 8 mm was
obtained (Figure 4a) under the conditions of the assay.
Electron microscopy images obtained after one hour in the
presence of 1e under lipid-catalyzed conditions confirmed the
much slower growth rate of fibers (Figure 4b,c). NMR-
binding experiments with rat IAPP, which does not form
fibers, showed a discrete binding interaction between 1e and
IAPP with an approximate Kd value of about 40 mm (see the
Supporting Information).
For both series of compounds, a comparison of the relative
t50 values for the lipid-catalyzed aggregation showed a gradual
increase in antagonistic activity from the dimer to the longer
oligomers, whereas the monomer had virtually no effect on
the kinetics (Figure 5a). The activities of the tetramers and
the pentamers were comparable although the pentamers were
slightly less active in both cases.
Angew. Chem. Int. Ed. 2010, 49, 736 –739
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
737

Communications
Figure 3. Relative rate of hIAPP (10 mm) aggregation in the presence of
1e (100 mm) compared to the control reaction showing: a) inhibition
under lipid-catalyzed and b) acceleration under lipid-free conditions.
Figure 5. Bar charts showing comparison of the relative rates of fiber
formation with the designed molecules; [hIAPP]=10 mm. a) Inhibition
under lipid-catalyzed conditions: DOPG/DOPC=1:1 (500 mgmLÀ1);
[compound]=150 mm, and b) acceleration under lipid-free conditions:
[compound]=10 mm.
conditions the compounds do not effectively partition into the
membrane while IAPP is bound at the membrane surface.
Diethylene triamine pentaacetic acid (DTPA; 3), a
control molecule bearing five carboxylic acid groups in a
less well-defined orientation, had very little effect on the
kinetics of aggregation (Figure 5). Whereas the lipid-cata-
lyzed kinetic data were virtually indistinguishable from the
control, DTPA accelerated the de novo fiber-formation
kinetics somewhat, perhaps as a result of nonspecific charge
neutralization effects. Control molecules bearing positively
charged (based on 1c[22]) or tert-butyl ester protected (1cester
and 1eester; see the Supporting Information for structures) side
chains also had little effect on lipid-catalyzed kinetics of
aggregation (see the Supporting Information). The lipid-free
kinetics for 1cester and 1eester, however, were not directly
comparable to 1e presumably because of their hydrophobic-
ity and highly aggregating nature. Taken together these data
suggest that the number, nature, and orientation of the
charges are crucial to the activity of the molecules, indicating
a specific interaction most likely with the complementary a-
helical region of IAPP. A detailed mechanistic study will be
reported elsewhere.[23]
Figure 4. a) Dose response curve for lipid-catalyzed IAPP amyloid
inhibition with 1e. Electron microscopy pictures showing: b) the
presence of fiber formation in the control reaction and c) absence of
fiber formation with 1e under lipid-catalyzed conditions after 1 h.
To probe the mechanism of inhibition of aggregation,
kinetic experiments were performed in the absence of the
lipid. Under these conditions, the molecules acted as agonists
of amyloid formation and compound 1e showed two- to
three-fold acceleration in the aggregation kinetics (Fig-
ure 3b). As with the lipid-catalyzed conditions, the rate of
acceleration was proportional to the length and charge of the
compounds, and the pentamers in both the series were the
most effective accelerants (Figure 5b). However, under lipid-
catalyzed conditions, higher bulk concentrations of the
compounds were required to strongly affect fiber formation
(Figure 5a versus Figure 5b) presumably because under these
In conclusion, two series of compounds based on an
oligoamide backbone were designed to provide a comple-
738
www.angewandte.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 736 –739

Angewandte
Chemie
Rev. Neurosci. 2003, 26, 267 – 298. J. R. Brender, K. Hartman,
K. R. Reid, R. T. Kennedy, A. Ramamoorthy, Biochemistry
2008, 47, 12680 – -12688.
mentary surface to interact with the a-helical domain on
IAPP. These molecules project their anionic substituents at
the right distance and orientation, and under lipid-free
conditions accelerate the aggregation of IAPP. Under lipid-
catalyzed conditions, however, they retard the formation of
amyloid deposits. Whereas both series of compounds follow
the same general trend, the oligopyridylamide series shows a
slight, but consistently higher effect potentially resulting from
a reduced entropic penalty upon binding (see the Supporting
Information). This study suggests the targeting of discrete
amyloidogenic intermediates as an alternative therapeutic
approach to amyloid diseases and paves the way for research
into novel type II diabetes drugs having a particular focus
upon inhibiting the lipid-catalyzed acceleration of IAPP
aggregation.
[11] R. Mishra, B. Bulic, D. Sellin, S. Jha, H. Waldmann, R. Winter,
Angew. Chem. 2008, 120, 4757 – 4760; Angew. Chem. Int. Ed.
2008, 47, 4679 – 4682.
[12] Y. Porat, Y. Mazor, S. Efrat, E. Gazit, Biochemistry 2004, 43,
14454 – 14462.
[13] M. Levy, Y. Porat, E. Bacharach, D. E. Shaley, E. Gazit,
Biochemistry 2008, 47, 5896 – 5904.
[14] J. E. Gestwicki, G. R. Crabtree, I. A. Graef, Science 2004, 306,
865 – 869; J. F. Aitken, K. M. Loomes, B. Konarkowska, G. J. S.
Cooper, Biochem. J. 2003, 374, 779 – 784.
[15] Y. Porat, A. Abramowitz, E. Gazit, Chem. Biol. Drug Des. 2006,
67, 27 – 37; E. Gazit, FASEB J. 2002, 16, 77 – 83.
[16] H. Yin, A. D. Hamilton, Angew. Chem. 2005, 117, 4200 – 4235;
Angew. Chem. Int. Ed. 2005, 44, 4130 – 4163 and references
therein; J. M. Davis, L. K. Tsou, A. D. Hamilton, Chem. Soc.
Rev. 2007, 36, 326 – 334; J. Becerril, A. D. Hamilton, Angew.
Chem. 2007, 119, 4555 – 4557; Angew. Chem. Int. Ed. 2007, 46,
4471 – 4473; J. M. Rodriguez, A. D. Hamilton, Angew. Chem.
2007, 119, 8768 – 8771; Angew. Chem. Int. Ed. 2007, 46, 8614 –
8617.
[17] J. T. Ernst, J. Becerril, H. S. Park, H. Yin, A. D. Hamilton,
Angew. Chem. 2003, 115, 553 – 557; Angew. Chem. Int. Ed. 2003,
42, 535 – 539; L. A. Estroff, C. D. Incarvito, A. D. Hamilton, J.
Am. Chem. Soc. 2004, 126, 2 – 3.
Received: March 28, 2009
Revised: October 13, 2009
Published online: December 22, 2009
Keywords: a-helix mimetics · amyloid b-peptides · diabetes ·
.
helical structures · proteomimetics
[1] F. E. Cohen, J. W. Kelly, Nature 2003, 426, 905 – 909; J. W. Kelly,
N. Engl. J. Med. 2005, 352, 722 – 723.
[18] I. Saraogi, C. D. Incarvito, A. D. Hamilton, Angew. Chem. 2008,
120, 9837 – 9840; Angew. Chem. Int. Ed. 2008, 47, 9691 – 9694.
[19] L. A. Estroff, Ph. D. Dissertation, Yale University (New Haven),
2003.
[2] R. L. Hull, G. T. Westermark, P. Westermark, S. E. Kahn, J. Clin.
Endocrinol. Metab. 2004, 89, 3629 – 3643.
[3] A. E. Butler, J. Jang, T. Gurlo, M. D. Carty, W. C. Soeller, P. C.
Butler, Diabetes 2004, 53, 1509 – 1516.
[4] J. D. Knight, J. A. Hebda, A. D. Miranker, Biochemistry 2006,
45, 9496 – 9508.
[5] F. Chiti, C. M. Dobson, Annu. Rev. Biochem. 2006, 75, 333 – 366.
[6] J. D. Knight, A. D. Miranker, J. Mol. Biol. 2004, 341, 1175 – 1187.
[7] S. A. Jayasinghe, R. Langen, Biochemistry 2005, 44, 12113 –
12119; J. A. Williamson, A. D. Miranker, Protein Sci. 2007, 16,
110 – 117; M. Apostolidou, S. A. Jayasinghe, R. Langen, J. Biol.
Chem. 2008, 283, 17205 – 17210.
[20] J. Plante, F. Campbell, B. Malkova, C. Kilner, S. L. Warriner,
A. J. Wilson, Org. Biomol. Chem. 2008, 6, 138 – 146.
[21] J. M. Ahn, S. Y. Han, Tetrahedron Lett. 2007, 48, 3543 – 3547.
[22] J. Becerril, Ph. D. Dissertation, Yale University (New Haven),
2007.
[23] J. A. Hebda, I. Saraogi, M. Magzoub, A. D. Hamilton, A. D.
Miranker, Chem. Biol. 2009, 16, 943 – 950.
[24] X-ray structures: CCDC 725332 (tert-butyl ester of 1d) contains
the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
[8] J. A. Hebda, A. D. Miranker, Annu. Rev. Biophys. 2009, 38, 125 –
152.
[9] R. Tycko, Curr. Opin. Struct. Biol. 2004, 14, 96 – 103.
[10] J. Janson, R. H. Ashley, D. Harrison, S. McIntyre, P. C. Butler,
Diabetes 1999, 48, 491 – 498; B. Caughey, P. T. Lansbury, Annu.
Angew. Chem. Int. Ed. 2010, 49, 736 –739
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
739